Compositions and methods for altering second messenger signaling

ABSTRACT

The present disclosure provides, among other things, novel cyclic-GMP-AMP (cGAMP) analogs, mimics, mimetics and variants, and compositions and kits thereof; methods of using the compounds as described herein for treating cancer, and immune disease, disorders, or conditions; methods of using the compounds as described herein for modulating cGAS and STING; and methods of designing or characterizing a cGAS modulator.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. provisional patentapplication Nos. 61/817,269, filed Apr. 29, 2013, and 61/819,369, filedMay 3, 2013, the entire contents of each of which are herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

The importance of cyclic dinucleotides as bacterial second messengers iswell established, with cyclic di-GMP (c-di-GMP) now acknowledged as auniversal bacterial second messenger. This versatile molecule has beenshown to play key roles in cell cycle and differentiation, motility andvirulence, as well as in the regulation of biofilm formation anddispersion. Advances in our understanding of c-di-GMP has emerged withthe identification, structural characterization, and mechanisticunderstanding of the catalytic activities of the bacterial enzymesresponsible for the synthesis and degradation of this second messenger.Crystal structures of c-di-GMP in the free state and when bound toenzymes responsible for its synthesis and degradation have shown thatthis second messenger can adopt either monomeric or a dimericbis-intercalated folds. It appears that formation of c-(3′,5′)-di-GMPfrom two molecules of GTP occurs via a two-step reaction and formationof 3′,5′-phosphodiester linkages, with two molecules of pyrophosphate asbyproducts of the cyclization reaction. Moreover, multiple receptorstargeted by c-(3′,5′)-di-GMP and the diverse ways bacteria signalthrough this second messenger have been identified. Indeed, the field ofc-di-GMP study as a second messenger has grown immensely and yieldedmajor advances in our understanding of the physiology and mechanisms ofbacterial cyclic dinucleotide signaling over the last two and a halfdecades. In parallel studies, c-(3′,5′)-di-GMP-specific riboswitcheshave also been identified, including ones that are involved in cyclicdinucleotide-induced RNA splicing.

There is much interest currently towards gaining a molecular andfunctional understanding of innate immunity sensors of higher metazoansthat recognize nucleic acids in the cytoplasm and trigger type Iinterferon induction. Cytoplasmic dsDNA of pathogenic bacterial or viralorigin, and perhaps also displaced nuclear or mitochondrial DNAfollowing cellular stress, represent such a trigger. These eventsinvolving self-nucleic acid recognition in turn could trigger autoimmunediseases such as systemic lupus erythematosus and Sjögren syndrome.Indeed, in recent years many cytoplasmic DNA sensors have beenidentified, including DAI (DNA-dependent activator of IFN-regulatoryfactor), LRRFIP1 (leucine-rich repeat and flightless I interactingprotein 1), DDX41 (DEAD box polypeptide 41), and members of the HIN-200(hematopoietic interferon-inducible nuclear proteins) family such asAIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16).Molecular information is available on the HIN domain family as reflectedin structures of their complexes with dsDNA. A requirement for multiplesensors may be a reflection of distinctive cell-type specificactivities. Cytoplasmic detection of dsDNA activates stimulator ofinterferon genes (STING) in the cytoplasm, which in turn initiates acascade of events by first activating kinases IKK (IκB kinase) and TBK1(TANK-binding kinase 1), leading to phosphorylation and activation ofthe transcription factors NF-κB (nuclear factor κB) and IRF3 (interferonregulatory factor). These phosphorylated transcription factorstranslocate to the nucleus to target immune and inflammatory genesleading to the production of cytokines and type I interferons, therebytriggering the host immune response. Therefore, there is a need fortherapeutic agents to modulate the induction of interferon and otherrelevant components in these pathways.

SUMMARY OF THE INVENTION

The present invention provides, among other things, novel cyclic-GMP-AMP(cGAMP) analogs, mimics, mimetics and variants as described in moredetail below. These cGAMP compounds and compositions are, among otherthings, useful in the design of research tools, as a research tool, andas therapeutice modalities such as enzyme modulators including agonistsand antagonists of cGAS. The present invention also providescrystallographic data for cyclic-GMP-AMP synthase (cGAS). Thesecrystallographic data provide the basis for which to design modulators(agonists and antagonists) such as cGAMP compounds or small molecules,which are useful in the fields of research, therapeutics and/ordiagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-H. Structures of cGAMP Synthase (cGAS) in the Free State andBound to dsDNA. (A) 2.0 Å crystal structure of cGAS in the free state.The backbone of the protein in a ribbon representation is colored inlight gray. (B) 2.1 Å crystal structure of cGAS bound to a complementary16-bp DNA duplex (with one base 5′-overhang at each end). The proteinand DNA are colored in dark gray in the binary complex. (C) A schematicof intermolecular hydrogen bonds in the binary cGAS-DNA complex. (D)Superposed structures of cGAS in the free state (light gray) and in thecGAS-DNA complex (dark gray). (E, F) Large changes within the β-sheet(panel E) and catalytic pocket (panel F) segments on proceeding fromcGAS in the free state (light gray) to the binary complex with bound DNA(dark gray). (G) Narrow entrance to the catalytic pocket in thestructure of cGAS in the free state, with the protein in anelectrostatic representation. (H) Widened entrance to the catalyticpocket in the structure of the binary cGAS-DNA complex.

FIG. 2A-H. Structure of the ternary complex of cGAS, dsDNA and ATP. (A)2.4 Å crystal structure of the ternary complex of cGAS bound to dsDNAand ATP. The protein and dsDNA are shown in ribbon, with bound ATP in aspace-filling representation. (B) Superposed structures of the binarycomplex of cGAS and DNA and the ternary complex with added ATP. (C, D)Absence of changes in the backbone within the β-sheet (panel C) andcatalytic pocket (panel D) segments on proceeding from the binary cGASand dsDNA complex to the ternary complex with added ATP. (E, F) Twoalternate views of intermolecular contacts between ATP and catalyticpocket residues in the ternary complex. Two cations are shown asspheres, with hydrogen bonds shown by dashed lines. (G) 2Fo-Fc electrondensity map contoured at 1.2σ (light gray) and Fo-Fc map contoured at3.0σ (dark gray) of bound ATP, pair of cations and coordinating residuesin the catalytic pocket. This map contains some weak unaccounted forelectron density (dark gray). (H) View of bound ATP in a space-fillingrepresentation within the catalytic pocket, with the protein in anelectrostatic representation.

FIG. 3A-H. Structure of the Ternary Complex of cGAS, dsDNA with BoundProducts 5′-pppG(2′,5′)pG and 5′-pG(2′,5′)pA. (A) 1.9 Å crystalstructure of the ternary complex of cGAS bound to dsDNA and5′-pppG(2′,5′)pG. The protein and DNA are shown in ribbon, with bound5′-pppG(2′,5′)pG in a space-filling representation. (B, C) Two alternateviews of intermolecular contacts between 5′-pppG(2′,5′)pG and catalyticpocket residues in the ternary complex. Two cations are shown asspheres, with hydrogen bonds shown by dashed lines. (D) 2Fo-Fc electrondensity map contoured at 1.2σ of bound 5′-pppG(2′,5′)pG in the catalyticpocket of the ternary complex. (E) View of bound 5′-pppG(2′,5′)pG in aspace-filling representation within the catalytic pocket, with theprotein in an electrostatic representation. (F, G) Two alternate viewsof intermolecular contacts between 5′-pG(2′,5′)pA and catalytic pocketresidues in the 2.3 Å ternary complex of cGAS, dsDNA and GMP+ATP. (H)Superposition of structures of bound 5′-pppG(2′,5′)pG (dark gray) and5′-pG(2′,5′)pA (light gray).

FIG. 4A-H. Structure of the Ternary Complex of cGAS, DNA with BoundProduct c[G(2′,5′)pA(3′,5′)p]. (A) 2.3 Å crystal structure of theternary complex of cGAS bound to dsDNA and productc[G(2′,5′)pA(3′,5′)p]. The protein and DNA are shown in ribbon, withbound product c[G(2′,5′)pA(3′,5′)p] in a space-filling representation.(B, C) Two alternate views of intermolecular contacts between productc[G(2′,5′)pA(3′,5′)p] and catalytic pocket residues in the ternarycomplex. (D) 2Fo-Fc electron density map contoured at 1.2σ of boundc[G(2′,5′)pA(3′,5′)p] in the catalytic pocket of the ternary complex.(E) View of bound c[G(2′,5′)pA(3′,5′)p] in a space-fillingrepresentation positioned towards on end of the catalytic pocket, withthe protein in an electrostatic representation. (F) A view ofc[G(2′,5′)pA(3′,5′)p] highlighting the 2′,5′ linkage at the GpA step andthe 3′,5′ linkage at the ApG step. (G) Stacking of the G residue of5′-pG(2′,5′)pA on Tyr 421 in its ternary complex with cGAS and dsDNA.(H) Stacking of the A residue of c[G(2′,5′)pA(3′,5′)p] on Tyr 421 in itsternary complex with cGAS and dsDNA.

FIG. 5A-D. Characterization of c[G(2′,5′)pA(3′,5′)p] Formation by cGAS.Generation of c[G(2′,5′)pA(3′,5′)p] and linear products andintermediates were assayed by thin-layer chromatography (TLC) usingpurified recombinant truncated (A panel, amino acids 147-507, used incrystallization studies) and full-length cGAS (B-D panels, amino acids1-507). Long- and short-dashed lines indicate the origin and solventfronts, respectively. (A) A 45-nt dsDNA was incubated with cGAS (x-y) inreaction buffer containing indicated divalent cation (or EDTA) andα³²p-ATP and -GTP. Chemically synthesized cGAMP containing both 3′,5′linkages was co-spotted in every sample and its migration, visualized byUV, is indicated (dashed outlines). (B) cGAS was incubated with single(ss) or double (ds) stranded DNA, RNA, DNA/RNA duplex, or 8-oxoguanine(8-O-G) modified DNA of similar sequence and c[G(2′,5′)pA(3′,5′)p]formation was monitored using α³²p-ATP. (C) Mono- and di-phosphorylatedadenosine and guanosine were used as substrates to determine order ofc[G(2′,5′)pA(3′,5′)p] formation. Slow-migrating 2′,5′-linkedintermediate species when cGAS and dsDNA is incubated with α³²p-ATP andGMP (5′-pGpA) or GDP (5′-ppGpA). (D) dsDNA-dependent cGAMP reactionintermediates were visualized by using 2′ or 3′ dATP and dGTP. Slowmigrating intermediate species, corresponding to pppGpA (lane 1) orpppGpdA (lanes 2 and 3), are seen by changing TLC mobile phasecomposition. Intermediate species were confirmed using γ³²p-GTP.

FIG. 6A-C. Definitive Identification of c[G(2′,5′)pA(3′,5′)p] as theEnzymatic Product of cGAS. (A) UV 260 nm chromatographs of GTP, ATP,c[G(2′,5′)pA(2′,5′)p], c[G(3′,5′)pA(3′,5′)p], c[G(2′,5′)pA(3′,5′)p] andcGAS reaction (rxn, asterisk) solutions from reverse-phase HPLCanalyses. cGAS reaction samples were injected alone or with addition ofindicated reference standards. Shaded region shows the retention timecorresponding to the elution of c[G(2′,5′)pA(3′,5′)p]. (B) UV 260 nmchromatographs from HPLC analysis of the cGAS product obtained fromdissolved crystals when injected alone (top trace), or co-injected withc[G(2′,5′)pA(2′,5′)p] reference compound (middle trace). Additionalunidentified peaks were present in the dissolved crystal solution, butelute later. The three reference cGAMP compounds were co-injected due toa change (0.5 sec) in the retention time of c[G(2′,5′)pA(3′,5′)p] as aresult of applying the dissolved crystal solution to the column. (C) NMRspectra of the sugar H1′ proton region of three chemically synthesizedcGAMP reference compounds with the cGAS rxn in 99.9% D₂O in 10 mMK₂HPO₄—KH₂PO₄ (pH 6.6) buffer. The NMR spectrum of the cGAS rxncorresponds to c[G(2′,5′)pA(3′,5′)p] reference compound. The H1′ protonis a doublet (³J_(HH)=9 Hz) when the phosphate is attached to the2′-position, but a singlet when the phosphate is attached to the3′-position, reflecting the different puckers of the five-membered sugarring dependent on the position of the attached phosphate group.

FIG. 7A-D. Functional analysis of cGAS Mutants and the Model forTwo-step Generation of c[G(2′,5′)pA(3′,5′)p]. (A) Levels ofc[G(2′,5′)pA(3′,5′)p] formation by cGAS full-length wt and indicatedmutants were compared by TLC analysis. Long- and short-dashed linesindicate the origin and solvent fronts, respectively. (B, C) Expressionvectors of murine cGAS WT, or carrying single and multiple alaninemutations of DNA binding (panel B) and catalytic (panel C) residues weretransiently transfected into HEK 293 cells together with an IFN-β Glucreporter, and constitutive STING and Firefly luc expression plasmids. Inthis setting expressed cGAS is engaged in the cytosol by theco-transfected DNA plasmids. Gluc values were determined in triplicate,36 h after transfection, normalized to Firefly luc, and are shown asfold induction over control plasmid (as mean±s.e.m). Data in panels Band C are representative of 3-5 independent experiments for each mutant.(D) A schematic representation of a proposed model associated with atwo-step generation of c[G(2′,5′)pA(3′,5′)p] within the single catalyticpocket of cGAS. In this model, the first step involves formation of a5′-pppGpA intermediate followed by formation of c[G(2′,5′)pA(3′,5′)p].Note, also that the bound ligand is believed to undergo two flip-overson the pathway to c[G(2′,5′)pA(3′,5′)p] formation.

FIG. 8A-C. Sequence Alignment and Crystal Structure of cGAS in the FreeState and Comparison with Human OAS1. (A) Sequence alignment of cGASfrom mouse and human (construct used for structural studies) spanningamino acids 147 to 507 (C-terminus). The putative catalytic residues areindicated in boxes. (B) Two alternate views of the structure of cGAS inthe free state. The backbone of the protein is shown in a ribbonrepresentation and colored in light gray. (C) Stereo view of superposedstructures of cGAS (light gray) and human oligoadenylate synthetase 1(OAS1) (black; PDB: 1PX5) in the free state. The r.m.s.d betweenstructures is 4.1 Å.

FIG. 9A-F. Molecular Recognition Features in the Structure of cGAS Boundto dsDNA and Comparison with hOAS1 Bound to dsRNA and 2′-Datp. (A, B)Examples of intermolecular contacts between cGAS and dsDNA. Watermolecules are shown as black spheres, with hydrogen bonds are indicatedby dashed lines. We observe one sequence-specific hydrogen bond betweenthe side chain of Arg161 and the O2 carbonyl of T8 as shown in panel B.(C, D, E) Examples of conformational shifts on proceeding from cGAS inthe free state (light gray) to the binary complex with bound dsDNA(gray). A shift of 5.1 Å is observed in the β-sheet segment on complexformation (panel C). A long α-helix breaks into two segments, with onesegment moving towards the dsDNA on complex formation, including theside chain of Arg161, which moves by 9.2 Å (panel D). Several Tyr andLys residues within loop segments shift between 6.7 and 17.6 Å oncomplex formation (panel E). (F) Stereo view of the superposedstructures of the protein components of cGAS in the dsDNA bound state(light gray) and OAS1 in the dsRNA bound state plus 2′-dATP (black, PDB:4IG8). The r.m.s.d between structures is 3.2 Å. The dsDNA bound to cGASand dsRNA bound to OAS1 are omitted from depiction for clarity.

FIG. 10A-I. Structure of cGAS with 5′-pppG(2′,5′)pG in the CatalyticPocket of its Ternary Complex Formed upon Crystallization with GTP. (A)Superposed structures of the binary complex of cGAS with DNA (gray) andthe ternary complex with bound 5′-pppG(2′,5′)pG intermediate product(dark gray). (B, C) Minimal changes are observed in the backbone withinthe β-sheet (panel B) and catalytic pocket (panel C) segments onproceeding from the binary complex to the ternary complex with bound5′-pppG(2′,5′)pG. (D) Two alternate views of the bound 5′-pppG(2′,5′)pGin the catalytic pocket of the ternary complex. Mg²⁺ cations are shownas spheres. Note that the alignment of bound ligand is5′-pppG(syn)p(2′,5′)pG(anti). (E) Two alternate views of the omit Fo-Fcomit electron density map contoured at 3.0σ of bound 5′-pppG(2′,5′)pG inthe catalytic pocket of the ternary complex. (F) Two alternate views ofthe superposed structures of bound 5′-pppG(2′,5′)pG (gray) and ATP (darkgray) in their respective ternary complexes with cGAS and dsDNA. (G)Omit map recorded at 4σ identifying two bound cations in the structureof the ternary complex. (H, I) Octahedral coordination geometry aroundthe two bound cations in the structure of the ternary complex.

FIG. 11A-F. Structure of cGAS and c[G(2′,5′)pA(3′,5′)p] Bound in theCatalytic Pocket of the Ternary Complex Formed upon Crystallization withGTP+ATP. (A) Superposed structures of the binary complex of cGAS and DNA(gray) and the ternary complex with added GTP+ATP for which the boundproduct is c[G(2′,5′)pA(3′,5′)p] (dark gray) obtained fromcrystallization with ATP and GTP. (B, C) No conformational changesoccurred in the backbone within the β-sheet (panel B) and catalyticpocket (panel C) segments on proceeding from the binary complex to theternary complex with bound c[G(2′,5′)pA(3′,5′)p]. (D) Two alternateviews of the bound product cGAMP in the catalytic pocket of the ternarycomplex. Note that the bound ligand c[G(2′,5′)pA(3′,5′)p] revealed a2′,5′ phosphodiester linkage within the GpA step. Based on HPLCcomparison, the structure of c[G(2′,5′)pA(3′,5′)p] is shown with a 3′,5′linkage at the ApG step. Both G and A residues adopt anti alignments attheir glycosidic bonds. (E) Two alternate views of the Fo-Fc omitelectron density map contoured at 3.0σ of bound c[G(2′,5′)pA(3′,5′)p] inthe catalytic pocket of the ternary complex. (F) Two alternate views ofthe superposed structures of bound c[G(2′,5′)pA(3′,5′)p] (gray) and ATP(dark gray) in their respective ternary complexes with cGAS and dsDNA.

FIG. 12A-C. Thin-layer Chromatography (TLC) Conditions for MonitoringFormation of Cgamp. (A, B) Indicated nucleotides were spotted onhigh-performance silica gel TLC plates, resolved by various solventsystems, and visualized by UV. Two mobile phase conditions were used (Aand B). Solvent system 1 was used in the majority of experiments fordetection of c[G(2′,5′)pA(3′,5′)p], whereas solvent 2 was used for abetter separation of the mono and tri-phosphorylated linearintermediates. Dashed lines indicate the solvent fronts. (C) CalculatedRf values.

FIG. 13A-E. dsDNA-length and Nucleotide Requirements of cGAS Activity.(A) Full-length cGAS was incubated with equimolar or mass-normalizedquantities of 16-, 36-, or 45-nt dsDNA then assayed for cGAMP formation.Long- and short-dashed lines in panels A-C, indicate the origin andsolvent fronts, respectively. (B) Truncated (tr) and full-length (fl)cGAS was incubated with 45 bp dsDNA in reaction buffer containing thevarious indicated nucleotides. cGAS (tr) exhibits activity, albeit lessthan cGAS (fl). c[G(2′,5′)pA(3′,5′)p] forms using 2′-dATP, when 2′-dATPor GTP was radiolabelled, but not at all when 2′-dGTP was used. 2′-dATPwith 2′-dGTP yielded no c[G(2′,5′)pA(3′,5′)p], indicating that blockageof the 2′ OH positions in adenosine, and more importantly guanosine,prevented c[G(2′,5′)pA(3′,5′)p] production. Asterisks (*) denote whichnucleotides were supplemented with an α³²p-radiolabelled form. dNTPindicates the triphosphorylated 2′-deoxynucleotide. (C) Full-length cGASwas incubated in reaction buffer containing dsDNA and the indicatedcombination of ribonucleotides, then analyzed by TLC. Trace amounts ofcyclic product were formed upon incubation of α³²p-ATP with UTP, orα³²p-GTP with GTP, CTP, and UTP. Optimal product formation requires GTPand ATP. The low level of cyclic product formation with UTP and ATP, butno ATP alone, suggests that UTP can be accommodated at the GTP bindingsite but reduced in affinity and/or activity. The migrations of allproducts are consistent with formation of cyclical dinucleotides. (D)HPLC analysis of dsDNA-dependent cGAS generation ofc[G(2′,5′)pA(3′,5′)p] over time. A single cGAS reaction was initiatedand samples were analyzed by HPLC at indicated times. (E) Highlyconserved residues G198 and S199 were mutated to alanine, or G198 toproline to reduce steric flexibility. Expression plasmids for mutant andWT cGAS were transiently transfected into HEK 293 cells together with anIFN-β Gluc reporter, and constitutive STING and Firefly luc expressionplasmids. Gluc values were determined in triplicate, 36 h aftertransfection, normalized to Firefly luc, and are shown as fold inductionover Control plasmid (as mean±s.e.m). Data are representative of 3independent experiments for each mutant.

FIG. 14A-B. Syntheses of cGAMP isomers. Synthesis of cGAMP containing2′,5′ linkages at both GpA and ApG steps (6) (top panel). Synthesis ofcGAMP containing 2′,5′ at GpA step and 3′,5′ at ApG step (11) (middlepanel). Synthesis of cGAMP containing 3′,5′ linkages at both GpA and ApGsteps (15) (bottom panel).

FIG. 15A-D. Resonance assignments of c[G(2′,5′)pA(3′,5′)p] from HMBC,COSY, and HSQC two-dimensional NMR spectra. (A) HMBC spectrum showingcorrelations between aromatics and the sugar C1′-H1′. (B) HMBC spectrumshowing correlations within sugar rings. In (A) and (B), correlationswithin the guanine base are connected by solid lines and assignments arespecified on the upper and left edges for protons and carbonsrespectively, while correlations within the adenine base are connectedby dashed lines and assignments are specified on the lower and rightedges for protons and carbons respectively; The large unsuppressed1-bond C—H couplings are indicated by blue lines connecting the coupledpairs of signals. (C) Double quantum filtered COSY spectrum. Guaninecorrelations are connected by solid lines and resonances are labeled onthe cross peaks above the diagonal; Adenine correlations are connectedby dashed lines and resonances are labeled on the cross peaks below thediagonal. (D) Aliphatic HSQC spectrum summarizing sugar proton andcarbon assignments. See also FIG. 6C and Table S4.

FIG. 16A-D. STING-dependent induction of murine alpha-interferon andhuman CXCL10 by cGAMP compounds. The dose-dependent biologicalactivities of indicated cGAMP isomers were measured by enzyme-linkedimmunosorbent assay (ELISA), quantifying for the induction of endogenousmurine α-interferon (m-Ifna) or human CXCL10 (h-CXCL10) proteins. (A-B),Mouse bone marrow derived macrophages (BMDM) were either treated firstwith Digitonin (Dig) to permeabilize plasma membranes prior to cGAMPaddition (A) or cGAMP isomers were passively delivered to cells byaddition in culture medium (B). (C), cGAMP activation was also measuredin human THP-1 cells. Data are representative of 2 independentexperiments, each done in triplicate (error bars, s.e.m.). (D), The halfmaximal effective concentration (EC₅₀) values were estimated based on4-parametric sigmoidal dose-response curves; 95% confidence intervalranges (CI) are provided.

FIG. 17 is an exemplary block diagram of a computing device and a mobilecomputing device.

FIG. 18 is an exemplary block diagram of a network environment forestablishing a multi-channel context aware communication environment.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

It has been shown that VSP-1 (Vibrio 7^(th) pandemic island-1) genesencode a novel class of dinucleotide cyclases, which preferentiallysynthesize a cyclic-GMP-AMP (designated cGAMP) molecule, therebyexpanding our horizon to cyclic GA-dinucleotides (Davies et al. 2012).More recently, cyclic GMP-AMP synthase (cGAS, official human gene symbolMB21D1) was identified as a cytoplasmic DNA sensor that activated thetype I interferon pathway by synthesizing the second messenger cGAMP(Sun et al. 2013; Wu et al. 2013). cGAS was shown to be a member of thenucleotidyltransferase family, and to be capable of generating a cGAMPin vitro from GTP and ATP in the presence of dsDNA (but not dsRNA),while chemically synthesized cGAMP containing a pair of 3′,5′ linkageswas shown to stimulate the production of interferon in THP1 and Raw264.7cells at concentrations as low as 10 nM. The authors also demonstratedthrough experiments involving either overexpression or knockdown ofcGAS, that the synthetic cGAMP bound to and activated STING, resultingin the activation of transcription factor IRF3 and subsequent inductionof interferon (Sun et al. 2013; Wu et al. 2013).

A critical assumption in these studies on cGAS was that cGAMP containeda pair of 3′,5′ linkages (Sun et al. 2013; Wu et al. 2013), in line withthose reported previously for c-di-GMP in bacterial systems as outlinedabove. The present invention encompasses the recognition that thepreviously assigned structure of cGAMP by Sun and Wu was incorrect.Thus, one aspect of the present invention is the identification of thepreviously unknown problem of misidentification of the structure ofcGAMP. The present disclosure combines structural, chemical, in vitrobiochemical and in vivo cellular assays to establish unequivocally thatthis second messenger unexpectedly contains 2′,5′ linkage at the GpAstep and 3′,5′ linkage at the ApG step {designatedc[G(2′,5′)pA(3′,5′)p]}, thus identifying correctly and for the firsttime, the founding member of a new family of metazoan second messengersregulating type I interferon induction in response to cytoplasmic DNA.

In certain embodiments, the present invention provides compoundscomprising cyclic GA-dinucleotides [c[G(2′,5′)pA(3′,5′)p]] containing a2′,5′ linkage (at the GpA step). In some embodiments, such compounds areuseful for the study of cellular signaling and immune surveillance inmetazoans. In some embodiments, such compounds are useful in thetreatment, diagnosis or prophylaxis of disorders, diseases or conditionsin medicine. In some embodiments, such compounds are useful to modulatetargets involved in immune response. In some embodiments, the compoundsand/or compositions of the invention are useful as research tools and/orreagents, particularly in kits and assays for biological or chemicalresearch.

The present invention also provides crystallographic data useful in thedesign of modulators of cGAS. In certain embodiments, the inventionprovides modulators of cGAS that comprises features to form appropriatebinding interactions with cGAS. In some embodiments, such modulatorscomprise features that form appropriate binding interactions withtargets that bind to cGAMP.

Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

Provided compounds may comprise one or more saccharide moieties. Unlessotherwise specified, both D- and L-configurations, and mixtures thereof,are within the scope of the disclosure. Unless otherwise specified, bothα- and β-linked embodiments, and mixtures thereof, are contemplated bythe present disclosure.

If, for instance, a particular enantiomer of a compound of the presentdisclosure is desired, it may be prepared by asymmetric synthesis,chiral chromatography, or by derivation with a chiral auxiliary, wherethe resulting diastereomeric mixture is separated and the auxiliarygroup cleaved to provide the pure desired enantiomers. Alternatively,where the molecule contains a basic functional group, such as amino, oran acidic functional group, such as carboxyl, diastereomeric salts areformed with an appropriate optically-active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers.

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from an aliphatic moietycontaining between one and six carbon atoms by removal of a singlehydrogen atom. Unless otherwise specified, alkyl groups contain 1-12carbon atoms. In certain embodiments, alkyl groups contain 1-8 carbonatoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms. Insome embodiments, alkyl groups contain 1-5 carbon atoms, in someembodiments, alkyl groups contain 1-4 carbon atoms, in some embodimentsalkyl groups contain 1-3 carbon atoms, and in some embodiments alkylgroups contain 1-2 carbon atoms. Examples of alkyl radicals include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl,dodecyl, and the like.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain aliphatic moiety having at least onecarbon-carbon double bond by the removal of a single hydrogen atom.Unless otherwise specified, alkenyl groups contain 2-12 carbon atoms. Incertain embodiments, alkenyl groups contain 2-8 carbon atoms. In certainembodiments, alkenyl groups contain 2-6 carbon atoms. In someembodiments, alkenyl groups contain 2-5 carbon atoms, in someembodiments, alkenyl groups contain 2-4 carbon atoms, in someembodiments alkenyl groups contain 2-3 carbon atoms, and in someembodiments alkenyl groups contain 2 carbon atoms. Alkenyl groupsinclude, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,and the like.

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain aliphatic moiety having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom.Unless otherwise specified, alkynyl groups contain 2-12 carbon atoms. Incertain embodiments, alkynyl groups contain 2-8 carbon atoms. In certainembodiments, alkynyl groups contain 2-6 carbon atoms. In someembodiments, alkynyl groups contain 2-5 carbon atoms, in someembodiments, alkynyl groups contain 2-4 carbon atoms, in someembodiments alkynyl groups contain 2-3 carbon atoms, and in someembodiments alkynyl groups contain 2 carbon atoms. Representativealkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl, and the like.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine

The term “halogen” means F, Cl, Br, or I.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to 10 ring members, whereinat least one ring in the system is aromatic and wherein each ring in thesystem contains three to seven ring members. The term “aryl” may be usedinterchangeably with the term “aryl ring”. In some embodiments, an 8-10membered bicyclic aryl group is an optionally substituted naphthyl ring.In certain embodiments of the present invention, “aryl” refers to anaromatic ring system which includes, but not limited to, phenyl,biphenyl, naphthyl, anthracyl and the like, which may bear one or moresubstituents. Also included within the scope of the term “aryl,” as itis used herein, is a group in which an aromatic ring is fused to one ormore non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 it electrons shared in a cyclic array; and having,in addition to carbon atoms, from one to five heteroatoms. Heteroarylgroups include, without limitation, thienyl, furanyl, pyrrolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”,as used herein, also include groups in which a heteroaromatic ring isfused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherethe radical or point of attachment is on the heteroaromatic ring.Nonlimiting examples include indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa 5-, 6- or 7-membered ring, unless otherwise specified, containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. The 5-membered ring has zeroto two double bonds, and the 6- and 7-membered rings have zero to threedouble bonds. Exemplary unsubstituted heterocyclyl groups are of 1 to 12(e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9)carbons. The term “heterocyclyl” also represents a heterocyclic compoundhaving a bridged multicyclic structure in which one or more carbonsand/or heteroatoms bridges two non-adjacent members of a monocyclicring, e.g., a quinuclidinyl group. The term “heterocyclyl” includesbicyclic, tricyclic, and tetracyclic groups in which any of the aboveheterocyclic rings is fused to one, two, or three carbocyclic rings,e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, acyclopentane ring, a cyclopentene ring, or another monocyclicheterocyclic ring, such as indolyl, quinolyl, isoquinolyl,tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples offused heterocyclyls include tropanes and1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may, when specified,contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(o); —(CH₂)₀₋₄OR^(o); —O(CH₂)₀₋₄R^(o), —O—(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄CH(OR^(o))₂; —(CH₂)₀₋₄SR^(o); —(CH₂)₀₋₄Ph, which may besubstituted with R^(o); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(o); —CH═CHPh, which may be substituted with R^(o);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(o); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(o))₂; —(CH₂)₀₋₄N(R^(o))C(O)R^(o);—N(R^(o)C(S)R^(o); —(CH₂)₀₋₄N(R^(o))C(O)NR^(o) ₂; —N(R^(o))C(S)NR^(o) ₂;—(CH₂)₀₋₄N(R^(o)) C(O)OR^(o); —N(R^(o))N(R^(o))C(O)R^(o);—N(R^(o))N(R^(o))C(O)NR^(o) ₂; —N(R^(o))N(R^(o))C(O)OR^(o);—(CH₂)₀₋₄C(O)R^(o); —C(S)R^(o); —(CH₂)₀₋₄C(O)OR^(o);—(CH₂)₀₋₄C(O)SR^(o); —(CH₂)₀₋₄C(O)OSiR^(o) ₃; —(CH₂)₀₋₄OC(O)R^(o);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(o); —(CH₂)₀₋₄SC(O)R^(o); —(CH₂)₀₋₄C(O)NR^(o)₂; —C(S)NR^(o) ₂; —C(S)SR^(o); —SC(S)SR^(o), —(CH₂)₀₋₄OC(O)NR^(o) ₂;—C(O)N(OR^(o))R^(o); —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o);—C(NOR^(o))R^(o); —(CH₂)₀₋₄SSR^(o); —(CH₂)₀₋₄S(O)₂R^(o);—(CH₂)₀₋₄S(O)₂OR^(o); —(CH₂)₀₋₄OS(O)₂R^(o); —S(O)₂NR^(o) ₂;—(CH₂)₀₋₄S(O)R^(o); —N(R^(o))S(O)₂NR^(o) ₂; —N(R^(o))S(O)₂R^(o);—N(OR^(o))R^(o); —C(NH)NR^(o) ₂; —P(O)₂R^(o); —P(O)R^(o) ₂; —OP(O)R^(o)₂; —OP(O)(OR^(o))₂; SiR^(o) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(o))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(o))₂, wherein each R^(o) may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(o), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(o) (or the ring formed by takingtwo independent occurrences of R^(o) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄straight or branched alkylene)C(O)OR^(•), or —SRR^(•) wherein each R^(•)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(o) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R^(*) ₂))₂₋₃O—,or —S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* isselected from hydrogen, C₁ aliphatic which may be substituted as definedbelow, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents thatare bound to vicinal substitutable carbons of an “optionallysubstituted” group include: —O(CR*₂)₂₋₃O—, wherein each independentoccurrence of R* is selected from hydrogen, C₁₋₆ aliphatic which may besubstituted as defined below, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(•) include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN,—C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein eachR^(•) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In another aspect, the present disclosure provides “pharmaceuticallyacceptable” compositions, which comprise a therapeutically effectiveamount of one or more of the compounds described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail, the pharmaceuticalcompositions of the present disclosure may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to theskin, lungs, or oral cavity; intravaginally or intrarectally, forexample, as a pessary, cream or foam; sublingually; ocularly;transdermally; or nasally, pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

In certain embodiments, neutral forms of the compounds are regeneratedby contacting the salt with a base or acid and isolating the parentcompound in the conventional manner. In some embodiments, the parentform of the compound differs from the various salt forms in certainphysical properties, such as solubility in polar solvents.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is masked or blocked,permitting, if desired, a reaction to be carried out selectively atanother reactive site in a multifunctional compound. Suitable protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. In certain embodiments, a protectinggroup reacts selectively in good yield to give a protected substratethat is stable to the projected reactions; the protecting group ispreferably selectively removable by readily available, preferablynon-toxic reagents that do not attack the other functional groups; theprotecting group forms a separable derivative (more preferably withoutthe generation of new stereogenic centers); and the protecting groupwill preferably have a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogen,and carbon protecting groups may be utilized. By way of non-limitingexample, hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 3,4-dimethoxybenzylidene acetal,2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethyleneacetal, α-methoxybenzylidene ortho ester,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described by Greene and Wuts (supra).

The symbol “

”, except when used as a bond to depict unknown or mixedstereochemistry, denotes the point of attachment of a chemical moiety tothe remainder of a molecule or chemical formula.

As used herein, the term “isolated” refers to a substance and/or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting), and/or (2) designed, produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. In some embodiments, as willbe understood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. In some embodiments, isolationinvolves or requires disruption of covalent bonds (e.g., to isolate apolypeptide domain from a longer polypeptide and/or to isolate anucleotide sequence element from a longer oligonucleotide or nucleicacid).

The term “modulator” is used to refer to an entity whose presence in asystem in which an activity of interest is observed correlates with achange in level and/or nature of that activity as compared with thatobserved under otherwise comparable conditions when the modulator isabsent. In some embodiments, a modulator is an activator or agonist, inthat activity is increased in its presence as compared with thatobserved under otherwise comparable conditions when the modulator isabsent. In some embodiments, a modulator is an inhibitor or antagonist,in that activity is reduced in its presence as compared with otherwisecomparable conditions when the modulator is absent. In some embodiments,a modulator interacts directly with a target entity whose activity is ofinterest. In some embodiments, a modulator interacts indirectly (i.e.,directly with an intermediate agent that interacts with the targetentity) with a target entity whose activity is of interest. In someembodiments, a modulator affects level of a target entity of interest;alternatively or additionally, in some embodiments, a modulator affectsactivity of a target entity of interest without affecting level of thetarget entity. In some embodiments, a modulator affects both level andactivity of a target entity of interest, so that an observed differencein activity is not entirely explained by or commensurate with anobserved difference in level. As used herein, an “activity” is anyprocess, carried out by a molecule, compound, cell, tissue or organ.Such processes may be catalytic or non-catalytic. For example, the cGASmolecules of the present invention may act as enzymes and as such mayhave enzymatic activity.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprise a polymer of nucleotides. These polymersare often referred to as polynucleotides. Exemplary nucleic acids orpolynucleotides of the invention include, but are not limited to,ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, a-LNA having an a-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine.

As will be clear from context, in some embodiments, “nucleic acid”refers to individual nucleic acid residues (e.g., nucleotides and/ornucleosides); in some embodiments, “nucleic acid” refers to anoligonucleotide chain comprising individual nucleic acid residues. Insome embodiments, a “nucleic acid” is or comprises RNA; in someembodiments, a “nucleic acid” is or comprises DNA. In some embodiments,a nucleic acid is, comprises, or consists of one or more natural nucleicacid residues. In some embodiments, a nucleic acid is, comprises, orconsists of one or more nucleic acid analogs. In some embodiments, anucleic acid analog differs from a nucleic acid in that it does notutilize a phosphodiester backbone. For example, in some embodiments, anucleic acid is, comprises, or consists of one or more “peptide nucleicacids”, which are known in the art and have peptide bonds instead ofphosphodiester bonds in the backbone, are considered within the scope ofthe present invention. Alternatively or additionally, in someembodiments, a nucleic acid has one or more phosphorothioate and/or5′-N-phosphoramidite linkages rather than phosphodiester bonds. In someembodiments, a nucleic acid is, comprises, or consists of one or morenatural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine). In some embodiments, a nucleic acid is, comprises, orconsists of one or more nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases,intercalated bases, and combinations thereof). In some embodiments, anucleic acid comprises one or more modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) ascompared with those in natural nucleic acids. In some embodiments, anucleic acid has a nucleotide sequence that encodes a functional geneproduct such as an RNA or protein. In some embodiments, a nucleic acidincludes one or more introns. In some embodiments, nucleic acids areprepared by one or more of isolation from a natural source, enzymaticsynthesis by polymerization based on a complementary template (in vivoor in vitro), reproduction in a recombinant cell or system, and chemicalsynthesis. In some embodiments, a nucleic acid is at least 2(dinucleotide or dinucleoside), 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000 or more residues long.

The term “polypeptide”, as used herein, generally has its art-recognizedmeaning of a polymer of at least three amino acids, linked to oneanother by peptide bonds. In some embodiments, the term is used to referto specific functional classes of polypeptides, such as, for example,receptors, enzymes, signaling proteins, structural proteins, autoantigenpolypeptides, nicotinic acetylcholine receptor polypeptides, alloantigenpolypeptides, etc. For each such class, the present specificationprovides several examples of amino acid sequences of known exemplarypolypeptides within the class; in some embodiments, such knownpolypeptides are reference polypeptides for the class. In some instancesthe polypeptide encoded is smaller than about 50 amino acids and thepolypeptide is then termed a peptide. If the polypeptide is a peptide,it will be at least about 2, 3, 4, or at least 5 amino acid residueslong. Thus, polypeptides include gene products, naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer, or tetramer. They may also comprisesingle chain or multichain polypeptides such as antibodies or insulinand may be associated or linked. Most commonly disulfide linkages arefound in multichain polypeptides. The term polypeptide may also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid. In such embodiments, the term “polypeptide” refers to anymember of the class that shows significant sequence homology or identitywith a relevant reference polypeptide. In many embodiments, such memberalso shares significant activity with the reference polypeptide. Forexample, in some embodiments, a member polypeptide shows an overalldegree of sequence homology or identity with a reference polypeptidethat is at least about 30-40%, and is often greater than about 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreand/or includes at least one region (i.e., a conserved region, oftenincluding a characteristic sequence element) that shows very highsequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or99%. Such a conserved region usually encompasses at least 3-4 and oftenup to 20 or more amino acids; in some embodiments, a conserved regionencompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments,a useful polypeptide as described herein may comprise or consist of afragment of a parent polypeptide. In some embodiments, a usefulpolypeptide as described herein may comprise or consist of a pluralityof fragments, each of which is found in the same parent polypeptide in adifferent spatial arrangement relative to one another than is found inthe polypeptide of interest (e.g., fragments that are directly linked inthe parent may be spatially separated in the polypeptide of interest orvice versa, and/or fragments may be present in a different order in thepolypeptide of interest than in the parent), so that the polypeptide ofinterest is a derivative of its parent polypeptide.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” in the context of polypeptides is meant to include polypeptidevariants which differ by one or more amino acid alterations, e.g.,substitutions, additions or deletions of amino acid residues that stillmaintain one or more of the properties of the parent or startingpolypeptide.

As used herein, the term “protein” refers to a polypeptide (i.e., astring of at least two amino acids linked to one another by peptidebonds). Proteins may include moieties other than amino acids (e.g., maybe glycoproteins, proteoglycans, etc.) and/or may be otherwise processedor modified. Those of ordinary skill in the art will appreciate that a“protein” can be a complete polypeptide chain as produced by a cell(with or without a signal sequence), or can be a characteristic portionthereof. Those of ordinary skill will appreciate that a protein cansometimes include more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. Polypeptidesmay contain L-amino acids, D-amino acids, or both and may contain any ofa variety of amino acid modifications or analogs known in the art.Useful modifications include, e.g., terminal acetylation, amidation,methylation, etc. In some embodiments, proteins may comprise naturalamino acids, non-natural amino acids, synthetic amino acids, andcombinations thereof. The term “peptide” is generally used to refer to apolypeptide having a length of less than about 100 amino acids, lessthan about 50 amino acids, less than 20 amino acids, or less than 10amino acids. In some embodiments, proteins are antibodies, antibodyfragments, biologically active portions thereof, and/or characteristicportions thereof.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal, and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “palliative” refers to treatment that is focused on the reliefof symptoms of a disease and/or side effects of a therapeutic regimen,but is not curative.

The term “therapeutic agent” or “therapeutic modality” refers to anyagent that, when administered to a subject, has a therapeutic,diagnostic, and/or prophylactic effect and/or elicits a desiredbiological and/or pharmacological effect.

As used herein, the term “therapeutically effective amount” means anamount of a substance (e.g., a therapeutic agent, composition, and/orformulation) that elicits a desired biological response whenadministered as part of a therapeutic regimen. In some embodiments, atherapeutically effective amount of a substance is an amount that issufficient, when administered to a subject suffering from or susceptibleto a disease, disorder, and/or condition, to treat the disease,disorder, and/or condition. As will be appreciated by those of ordinaryskill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

As used herein, the term “treat,” “treatment,” or “treating” refers toany method used to partially or completely alleviate, ameliorate,relieve, inhibit, prevent, delay onset of, reduce severity of and/orreduce incidence of one or more symptoms or features of a disease,disorder, and/or condition. Treatment may be administered to a subjectwho does not exhibit signs of a disease, disorder, and/or condition. Insome embodiments, treatment may be administered to a subject whoexhibits only early signs of the disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

The expression “unit dose” as used herein refers to a physicallydiscrete unit of a formulation appropriate for a subject to be treated.It will be understood, however, that the total daily usage of aformulation of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular subject or organism may dependupon a variety of factors including the disorder being treated and theseverity of the disorder; activity of specific active compound employed;specific composition employed; age, body weight, general health, sex anddiet of the subject; time of administration, and rate of excretion ofthe specific active compound employed; duration of the treatment; drugsand/or additional therapies used in combination or coincidental withspecific compound(s) employed, and like factors well known in themedical arts. A particular unit dose may or may not contain atherapeutically effective amount of a therapeutic agent.

As used herein, the term “patient” or “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate) to whom therapy is administered. In manyembodiments, a patient is a human being. A human includes pre and postnatal forms. In some embodiments, a patient is a human presenting to amedical provider for diagnosis or treatment of a disease, disorder orcondition. In some embodiments, a patient displays one or more symptomsor characteristics of a disease, disorder or condition. In someembodiments, a patient does not display any symptom or characteristic ofa disease, disorder, or condition. In some embodiments, a patient issomeone with one or more features characteristic of susceptibility to orrisk of a disease, disorder, or condition.

As used herein, the term “sample” or “biological sample” refers to asubset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

As used herein “stable” refers to a compound or molecule that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent. Alternatively a compound or molecule maybe said to be stable if it is sufficiently robust to withstand anytreatment, insult or utilization without undergoing substantialdegradation prior to a selected timepoint, event or localization.

As used herein, the term “stabilize”, “stabilized,” “stabilized region”means to make or become stable.

An individual who is “suffering from” a disease, disorder, and/orcondition has been diagnosed with and/or displays one or more symptomsof the disease, disorder, and/or condition.

An individual who is “susceptible to” a disease, disorder, and/orcondition has not been diagnosed with the disease, disorder, and/orcondition. In some embodiments, an individual who is susceptible to adisease, disorder, and/or condition may exhibit symptoms of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will develop the disease, disorder, and/or condition.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition will not develop the disease, disorder,and/or condition.

The term “computer-readable medium”, as used herein, refers tonon-volatile (i.e. secondary storage) computer data storage and/ormemory to retain digital data even when not powered. Examples ofcomputer-readable medium include, but are not limited to hard disk,floppy disk, flash memory (i.e. solid state memory), Ferroelectric RAM(F-RAM), Magnetoresistive RAM (MRAM), optical disc, standalone RAMdisks, ZIP drives, magnetic tape and holographic memory.

The term “computer system” or “computer”, as used herein, refers to acomputing device that can be used to implement the techniques describedin this disclosure. An exemplary computing device 2500 and a mobilecomputing device are shown in FIG. 18.

As used herein, the term “crystal structure” of a composition shall meana computer readable medium in which is stored a representation of threedimensional positional information (i.e. coordinates) for atoms of thecomposition.

As used herein, the term “docking” refers to orienting, rotating,translating a chemical entity in the binding pocket, domain, molecule ormolecular complex or portion thereof based on distance geometry orenergy. Docking may be performed by distance geometry methods that findsets of atoms of a chemical entity that match sets of sphere centers ofthe binding pocket, domain, molecule or molecular complex or portionthereof. See Meng et al. J. Comp. Chem. 4: 505-524 (1992). Spherecenters are generated by providing an extra radius of given length fromthe atoms (excluding hydrogen atoms) in the binding pocket, domain,molecule or molecular complex or portion thereof. Real-time interactionenergy calculations, energy minimizations or rigid-body minimizations(Gschwend et al., J. Mol. Recognition 9:175-186 (1996)) can be performedwhile orienting the chemical entity to facilitate docking. For example,interactive docking experiments can be designed to follow the path ofleast resistance. If the user in an interactive docking experiment makesa move to increase the energy, the system will resist that move.However, if that user makes a move to decrease energy, the system willfavor that move by increased responsiveness. (Cohen et al., J. Med.Chem. 33:889-894 (1990)). Docking can also be performed by combining aMonte Carlo search technique with rapid energy evaluation usingmolecular affinity potentials. See Goodsell and Olson, Proteins:Structure, Function and Genetics 8:195-202 (1990). Software programsthat carry out docking functions include but are not limited to MATCHMOL(Cory et al., J. Mol. Graphics 2: 39 (1984); MOLFIT (Redington, Comput.Chem. 16: 217 (1992)) and DOCK (Meng et al., supra).

As used herein, the term “designed” refers to an agent (i) whosestructure is or was selected by the hand of man; (ii) that is producedby a process requiring the hand of man; and/or (iii) that is distinctfrom natural substances and other known agents.

As used herein, the term “storage environment” comprises any environmentcomprising secondary storage, i.e. long-term persistent storage. In someembodiments, a storage environment comprises computer-readable medium.In some embodiments, a storage environment comprises a networkenvironment for establishing a multi-channel context aware communicationenvironment (i.e. cloud computing). For example, FIG. 18 is a blockdiagram of a network environment for establishing a multi-channelcontext aware communication environment.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

Compounds and Compositions of the Invention

It has surprisingly been discovered, contrary to the disclosures in theart, that the founding member of a family of metazoan cyclicdinucleotide second messengers regulating type I interferon induction inresponse to cytoplasmic DNA comprises unique features heretofore notknown. These insights now afford the opportunity to design analogs,mimics, and/or mimetics of the molecule identified and to further usethe parent molecule or molecules designed based on the structure of theparent molecules in research and development.

cGAMP Analogs, Mimics, Mimetics and Modifications

The family of second messengers, termed cGAMPs or cyclic GAMPs, includesone or more of the cyclic structures defined as having at least oneguanine (G) and one adenine (A) nucleotide and being linked in a cyclicfashion. The linkages between the two nucleotides involve sugar tobackbone bond formation. According to the present invention, there arefour primary parent members of the cGAMP family. These include[c[G(2′,5′)pA(3′,5′)p]], [c[G(2′,5′)pA(2′,5′)p]],[c[G(3′,5′)pA(2′,5′)p]], [c[G(3′,5′)pA(3′,5′)p]]. Furthermore, each ofthe nucleotides of the pairs may adopt either syn or anti glycosidictorsion orientations.

As used herein, the term “cGAMP” refers to any of the parent moleculesof the family as well as any of the possible torsion orientations.Individual members of the family may be referred to by theirsugar-backbone linkage form, e.g., the newly discovered secondmessenger, [c[G(2′,5′)pA(3′,5′)p]], may be referred to as the“2-prime-3′prime” isomer, referencing the position on the sugar ringforming the sugar-backbone bond. The other isomers may be namedlikewise. Collectively, the compounds of the invention which are wildtype, analogs, mimics, mimetics or modified versions of the cGAMPfamily, are referred to as the group of “cGMP compounds.”

With the teachings provided herein, one of skill may now design analogs,mimics, or modifications to any of the parent molecules for use as amodulator, either agonist or antagonist, of the cGAS enzyme, andultimately as a modulator of downstream physiologic events associatedwith interferon signaling. Linear versions may also be designed aseither agonists, antagonists, or competitive inhibitors of cGAS ordownstream signaling events associated with either cGAS enzyme activityor interferon signaling.

The present invention contemplates several types of compounds orcompositions which are nucleic acid based including variants andderivatives. These include substitutional, insertional, deletion andcovalent variants and derivatives. The term “derivative” is usedsynonymously with the term “variant” but generally refers to a moleculethat has been modified and/or changed in any way relative to a referencemolecule or starting molecule.

As used herein, an “analog” is meant to include cGAMP variants whichdiffer by one or more alterations, e.g., substitutions, additions ordeletions that still maintain one or more of the properties of theparent or starting molecule. Analogs are typically designed usingstructure-activity relationships (SAR) such as those described herein.

cGAS Proteins, Variants, Derivatives and Mutants

Having now in hand several protein crystal structures in native andvarying binding states, it is possible to exploit these proteinstructures by designing variants, derivatives or mutants of the cGASenzyme. As such, cGAS enzyme polypeptides, including their variants,derivatives and mutants are considered compounds of the invention. Thesevariants, derivatives and mutants are useful as research tools, forexample in kits or assays or as the source of a therapeutic modality. Tothis end, fragments or portions of the cGAS polypeptide or the variants,derivatives and mutants may be used as antigens for the production ofantibodies, or where the fragment maintains a structural elementassociated with activity, whether binding, catalysis, or transport mayalso be used as a modulator of the enzyme itself or as a surrogate fordsDNA binding. Collectively, the compounds of the invention which arewild type, variants, derivatives or mutants of cGAS, are referred to asthe group of “cGAS molecules.” cGAS molecules may comprise any portionor fragment of a cGAS molecule or may comprise mixed domains orfragments from cGAS molecules arising from different structures asdefined by the crystal structures disclosed herein.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, cGAS encoding polypeptides containing substitutions,insertions, and/or additions, deletions and covalent modifications withrespect to reference sequences, in particular the polypeptide sequencesdisclosed herein, are included within the scope of this invention. Forexample, sequence tags or amino acids, such as one or more lysines, canbe added to the peptide sequences of the invention (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of the aminoacid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)) the contents of which are incorporated by reference it itsentirety.

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of the cGASpolypeptides encoded by the present invention include surfacemanifestations, local conformational shape, folds, loops, half-loops,domains, half-domains, sites, termini or any combination thereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide of the invention, any of severalmanipulations and/or modifications of these features may be performed bymoving, swapping, inverting, deleting, randomizing or duplicating.Furthermore, it is understood that manipulation of features may resultin the same outcome as a modification to the molecules of the invention.For example, a manipulation which involved deleting a domain wouldresult in the alteration of the length of a molecule just asmodification of a nucleic acid to encode less than a full lengthmolecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Antibodies

In some embodiments, the cGAMP compounds or cGAS molecules may be usedto generate antibodies. As such, the antibodies so generated areconsidered further compounds and compositions of the present invention.As used herein, term “antibody” includes monoclonal antibodies(including full length antibodies which have an immunoglobulin Fcregion), antibody compositions with polyepitopic specificity,multispecific antibodies (e.g., bispecific antibodies, diabodies, andsingle-chain molecules), as well as antibody fragments. The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein. Asused herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity. Chimericantibodies of interest herein include, but are not limited to,“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g., Old World Monkey, Apeetc.) and human constant region sequences.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies; nanobodies; single-chainantibody molecules and multispecific antibodies formed from antibodyfragments.

Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM,may be generated by the compounds or molecules of the invention,including the heavy chains designated alpha, delta, epsilon, gamma andmu, respectively.

While not wishing to be bound by theory, it is believed that antibodiesgenerated using the cGAMP compounds or cGAS molecules disclosed hereinwill result in improved therapeutic efficacy.

Antibodies of the invention may be utilized to treat conditions ordiseases in many therapeutic areas such as, but not limited to, blood,cardiovascular, CNS, poisoning (including antivenoms), dermatology,endocrinology, gastrointestinal, medical imaging, musculoskeletal,oncology, immunology, inflammation, respiratory, sensory andanti-infective.

In one embodiment, variants of antibodies may also include, but are notlimited to, substitutional variants, conservative amino acidsubstitution, insertional variants, deletional variants and/or covalentderivatives.

Vaccines

As used herein, a “vaccine” is a biological preparation that improvesimmunity to a particular disease or infectious agent. According to thepresent invention and while not wishing to be bound by theory, it isbelieved that utilization of the cGAMP compounds or cGAS molecules ofthe invention may be used as a vaccine or as vaccine adjuvant.

Vaccines of the invention may be utilized to treat conditions ordiseases in many therapeutic areas such as, but not limited to,cardiovascular, CNS, dermatology, endocrinology, oncology, immunologyand autoimmunity, inflammation, respiratory, and anti-infective.

ALB Compounds

In some embodiments, the present invention provides a modulator of apolypeptide that binds cGAMP having a structure comprising the followingfeatures:A-L-Bwherein:A is or comprises a moiety that fits in the cGAS adenosine binding site;B is or comprises a moiety that fits in the cGAS guanosine binding site;andoptionally, L is a linker moiety linking A and B in a manner whichallows A and B to adopt appropriate interactions to bind cGAS.

In some embodiments, the polypeptide that binds cGAMP is cGAS. In someembodiments, the polypeptide that binds cGAMP is STING.

In some embodiments, A is Ring A as defined below and described inclasses and subclasses herein, both singly and in combination. In someembodiments, A optionally makes one or more interactions with cGAS atone or more sites selected from the group consisting of Ser199, Ser420,Lys402, Glu211, Asp213, Asp307, Tyr421, Arg364, and combinationsthereof. In some embodiments, A optionally makes one or moreinteractions with cGAS at one or more sites selected from the groupconsisting of Tyr421, Asp213, Asp307, Arg364, and combinations thereof.

In some embodiments, B is Ring B as defined below and described inclasses and subclasses herein, both singly and in combination. In someembodiments, B optionally makes one or more interactions with cGAS atone or more sites selected from the group consisting of Tyr421, Thr197,Ser366, Ser368, Arg364, and combinations thereof.

In some embodiments, a linker moiety is a linker suitable to covalentlylink A and B and which allows A and B to adopt appropriate interactionsto bind cGAS. In some embodiments, a linker together with A and/or Bcomprises a nucleoside optionally containing one or more phosphategroups. In some embodiments, a linker together with A and B comprises acyclic dinucleoside optionally containing one or more phosphate groups.In some embodiments, a modulator is a cyclic-GMP-AMP analog.

In some embodiments, a linker moiety comprises one or more ribose orphosphate groups. In some embodiments, such ribose and phosphate groups,along with Ring A or B, form a ribonucleotide. In some embodiments, amodulator comprises one or more modified ribonucleotides. Modifiedribonucleotides are well known in the art, and include modifications toa phosphate group, ribose group, nucleotide base group, and combinationsthereof. The present invention contemplates all possible modifiedribonucleotides for modulators and compound described herein. In someembodiments, these modifications enhance compound stability in vivo. Insome embodiments, modifications increase compound resilience tophosphodiesterases.

In some embodiments, a linker comprises a modified phosphodiester group.Such modifications are known in the art and include, without limitation,substituting phosphodiesters with phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates,boranophosphates, and combinations thereof. In some embodiments, aphosphodiester is modified to a phosphoramidates. Suitablephosphoramidates include, without limitation, those listed available atwww.glenresearch.com/Reference/StructureListing.php, the entire contentsof which are hereby incorporated by reference.

In some embodiments, a linker does not include phosphorus. In someembodiments, a linker comprises a short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane moieties; sulfide, sulfoxide and sulfone moieties;amides, carboxylates, formacetyl and thioformacetyl moieties; methyleneformacetyl and thioformacetyl moieties; riboacetyl moieties; alkenecontaining moieties; sulfamate moieties; methyleneimino andmethylenehydrazino moieties; sulfonate and sulfonamide moieties; amidemoieties; and others having mixed N, O, S, and CH₂ component parts.

In addition, a phosphodiester linker may be modified to improve thestability of the compound. For example, in certain instances the P═Olinkage is changed to a P═S linkage which is not as susceptible todegradation by nucleases in vivo. In certain instances, the C-2 hydroxylgroup of the sugar moiety of a nucleotide is converted to an alkyl orheteroalky ether. This modification renders the oligonucleotide lessprone to nucleolytic degradation.

Additional phosphodiester modification are described by Dellinger et al.Curr Protoc Nucleic Acid Chem. 2004 October; Chapter 4: Unit 4; Marshallet al. Science. 1993 Mar. 12; 259(5101):1564-70, the entire contents ofwhich are hereby incorporated by reference.

A linker moiety may also comprise one or more modified ribose moieties.In some embodiments, a linker comprises a ribose modified at one of thefollowing at the 2′ or 3′ position: OH; F; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. In some embodiments, 2′ or 3′modifications include: 2′-O-Me, 2′-O-MOE, 2′-O-allyl,2′-O-dinitrophosphate, 2′-fluoro, 2′-thio, 2′-aminoethyl,2′-guanidinopropyl. In some embodiments, 2′ or 3′ modifications include:O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)CH₃]₂, where n and m are from 1 toabout 10. In some embodiments, a linker comprises a ribose modified atthe 2′ or 3′ position with: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA-cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.In some embodiments, a modification includes 2′-O-methoxyethyl(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or2′-methoxyethoxy or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78,486-504) i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, and 2′-dimethylamino-ethoxyethoxy (also known in theart as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE),2′-O—CH₂—O—CH₂—N(CH₃)₂. Other modifications to a linker ribose include2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl(2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The2′-modification may be in the arabino (up) position or ribo (down)position. In some embodiments, a 2′-arabino modification is 2′-F.

Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position. Oligonucleotides may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

In some embodiments, a linker ribose is modified to a Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—) n group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in International Patent PublicationNos. WO 98/39352 and WO 99/14226, the entire contents of which arehereby incorporated by reference. In some embodiments, the LNA forms amoiety:

In certain embodiments, a linker comprises a hexose moiety. In someembodiments, the hexose is glucose or mannose. In certain instances, theribose sugar moiety is replaced with a cyclohexenyl group or polycyclicheteroalkyl ring. In some embodiments, the ribose sugar moiety isreplaced with morpholino group. Additional ribose modification arediscussed by Engels, New Biotechnology, Vol. 30, 3, p. 302 (2013), theentire contents of which are hereby incorporated by reference.

In some embodiments A or B is an unmodified or natural nucleobaseselected from adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). In some embodiments, A or B isa modified nucleobase. Modified nucleobases are known in the art andinclude, without limitation, synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. In some embodiments, a non-natural nucleobase isdifluorotolyl, nitropyrrolyl, or nitroimidazolyl. In certainembodiments, a non-natural nucleobase is 7-deazaadenine, 3-deazaadenine,N1-methyl-guanosine, 6-thioguanosine, 2-pyrimidinone, 4-thiouridine,2-pyridinone, 5-propynyl-uridine, imidazole-4-carboxamide, 5-nitroindol,3-nitropyrrole, 2-aminopurine, 5-methyl-2-pyrimidinone,N3-thioethylthymidine, 6-thiopurine, 5-iodouridine, 8-azidoadenosine,5-mercaptouridine, or those derived from 5-bromouracil, diaminopurine,2-thiouracil, 4-thiouracil, pseudouracil, difluorotoluene, anddihydrouracil. Additional modified nucleobases include those found inwww.glenresearch.com/Reference/StructureListing.php andwww.thermoscientificbio.com/rna-pricing-and-modifications/, the entirecontents of each of which are hereby incorporated by reference.Additional modifications are discussed by Verma et al. Annu Rev.Biochem. 1998. 67:99-134.

In some embodiments, a linker moiety comprises a group that replacesboth a phosphodiester and ribose groups of a ribonucleotide. One suchlinker is referred to as a peptide nucleic acid (PNA). In PNA compounds,the usual sugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, for example an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500; Nielsen et al., Chem. Soc. Rev., 1997,26, 73-78; Shakeel et al., Journal of Chemical Technology &Biotechnology, Volume 81, Number 6, June 2006, pp. 892-899(8); Nielsen,CHEMISTRY & BIODIVERSITY—Vol. 7 (2010), p. 786.

These and other suitable linkers are discussed in U.S. Pat. Nos.7,365,058, 8,101,348, 8,088,902, 7,579,451, 7,582,744, 8,334,373,8,017,762, 7,919,612, 7,812,149, and 7,723,508, the entire contents ofeach of which are hereby incorporated by reference herein.

In some embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof,wherein:Ring A is selected from the group consisting of:

Ring B is selected from the group consisting of:

-   each X¹ and X² is independently —CR— or —N—;-   X³ is —C(R)₂—, —O—, or —NR—;-   X^(a) and X^(b) are independently —C(R)₂—, —C(R)═C(R)—, —O—, —S—,    —S(O)—, —S(O)₂—, or —N(R)—;-   X^(a1) and X^(b1) are independently —C(R)— or —N—;-   X^(c) and X^(d), when present, are independently optionally    substituted oxygen, optionally substituted sulfur, a substituted    nitrogen atom, BH₃, or optionally substituted C₁₋₁₂ aliphatic;-   each X^(e) and X^(f) is independently —O—, —S—, or —N(R)—;-   each W is independently P or S;-   each R¹ and R² is independently selected from the group consisting    of hydrogen, halogen, —NO₂, —CN, —OR^(a), —SR, —N(R)₂, —C(O)R,    —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R, —C(O)N(R)₂,    —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂, —N(R)C(═NR)N(R)₂,    —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂, —N(R)SO₂R,    —OC(O)N(R)₂, and optionally substituted C₁₋₁₂ aliphatic or C₁₋₄    alkoxy-C₁₋₄ alkyl;-   each R³, R⁴, R⁵, R⁶, R⁷, R¹⁰, and R¹¹ is independently selected from    the group consisting of hydrogen, halogen, —NO₂, —CN, —OR, —SR,    —N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,    —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,    —N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂,    —N(R)SO₂N(R)₂, —N(R)SO₂R, —OC(O)N(R)₂, or an optionally substituted    group selected from C₁₋₁₂ aliphatic, phenyl, a 3-7 membered    saturated or partially unsaturated monocyclic carbocyclic ring, a    7-10 membered saturated or partially unsaturated bicyclic    carbocyclic ring, a 3-7 membered saturated or partially unsaturated    heterocyclic ring having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially    unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, and a 5-6    membered heteroaryl ring having 1-3 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   each R⁸ and R⁹, when present, is independently selected from the    group consisting of hydrogen, halogen, —NO₂, —CN, —OR^(a), —SR,    —N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,    —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,    —N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂,    —N(R)SO₂N(R)₂, —N(R)SO₂R, —OC(O)N(R)₂, and an optionally substituted    C₁₋₁₂ aliphatic;-   each R is independently selected from the group consisting of    hydrogen or an optionally substituted group selected from C₁₋₆    aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated    carbocyclic ring, a 3-7 membered saturated or partially unsaturated    monocyclic heterocyclic ring having 1-2 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, and a 5-6 membered    heteroaryl ring having 1-3 heteroatoms independently selected from    nitrogen, oxygen, or sulfur; or:-   two R groups on the same nitrogen are taken together with their    intervening atoms to form an optionally substituted 3-7 membered    saturated, partially unsaturated, or heteroaryl ring having 1-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur;    and-   R^(a) is an oxygen protecting group or R.

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring B is

In some embodiments, Ring B is

In some embodiments, X¹ is —CR—. In some embodiments, X¹ is —N—. In someembodiments, X² is —CR—. In some embodiments, X² is —N—. In someembodiments, X³ is —C(R)₂—. In some embodiments, X³ is —O—. In someembodiments, X³ is —NR—.

In certain embodiments, X^(a) is —C(R)₂—. In certain embodiments, X^(a)is —C(R)═C(R)—. In certain embodiments, X^(a) is —O—. In certainembodiments, X^(a) is —S—. In certain embodiments, X^(a) is —S(O)—.

In certain embodiments, X^(a) is —S(O)₂—. In certain embodiments, X^(a)is —NR—.

In certain embodiments, X^(b) is —C(R)₂—. In certain embodiments, X^(b)is —C(R)═C(R)—. In certain embodiments, X^(b) is —O—. In certainembodiments, X^(b) is —S—. In certain embodiments, X^(b) is —S(O)—. Incertain embodiments, X^(b) is —S(O)₂—. In certain embodiments, X^(b) is—NR—.

In certain embodiments, X^(a1) is —C(R)—. In certain embodiments, X^(a1)is —N—. In certain embodiments, X^(b1) is —C(R)—. In certainembodiments, X^(b1) is —N—.

In some embodiments, X^(c) is oxygen. In some embodiments, X^(c) issulfur. It will be appreciated that in certain embodiments where X^(c)is oxygen or sulfur, the oxygen or sulfur atom may possess a formalnegative charge. In some embodiments, X^(c) is a substituted nitrogenatom. In some embodiments, the nitrogen is independently substitutedwith hydrogen or optionally substituted C₁₋₁₂ aliphatic groups. In someembodiments, X^(c) is optionally substituted C₁₋₁₂ aliphatic.

In some embodiments, X^(d) is oxygen. In some embodiments, X^(d) issulfur. It will be appreciated that in certain embodiments where X^(d)is oxygen or sulfur, the oxygen or sulfur atom may possess a formalnegative charge. In some embodiments, X^(d) is a substituted nitrogenatom. In some embodiments, the nitrogen is independently substitutedwith hydrogen or optionally substituted C₁₋₁₂ aliphatic groups. In someembodiments, X^(d) is optionally substituted C₁₋₁₂ aliphatic.

In some embodiments, X^(e) is —O—. In some embodiments, X^(e) is —S—. Insome embodiments, X^(e) is —N(R)—.

In some embodiments, X^(f) is —O—. In some embodiments, X^(f) is —S—. Insome embodiments, X^(f) is —N(R)—.

In some embodiments, W is P. In other embodiments, W is S.

In some embodiments, R¹ is hydrogen, halogen, —OR^(a), —SR, —N(R)₂, andoptionally substituted C₁₋₁₂ aliphatic or C₁₋₄ alkoxy-C₁₋₄ alkyl. Insome embodiments, R¹ is hydrogen. In some embodiments, R¹ is halogen. Insome embodiments, R¹ is —OR^(a). In some embodiments, R¹ is —OH. In someembodiments, R¹ is fluro. In some embodiments, R¹ is C₁₋₁₂ aliphatic. Insome embodiments, R¹ is C₁₋₆ aliphatic. In some embodiments, R¹ is C₁₋₃aliphatic. In some embodiments, R¹ is methyl. In some embodiments, R¹ isC₁₋₄ alkoxy-C₁₋₄ alkyl. In some embodiments, R¹ is methoxy-ethyl.

In some embodiments, R² is hydrogen, halogen, —OR^(a), —SR, —N(R)₂, andoptionally substituted C₁₋₁₂ aliphatic or C₁₋₄ alkoxy-C₁₋₄ alkyl. Insome embodiments, R² is hydrogen. In some embodiments, R² is halogen. Insome embodiments, R² is —OR^(a). In some embodiments, R² is —OH. In someembodiments, R² is fluro. In some embodiments, R² is C₁₋₁₂ aliphatic. Insome embodiments, R² is C₁₋₆ aliphatic. In some embodiments, R² is C₁₋₃aliphatic. In some embodiments, R² is methyl. In some embodiments, R² isC₁₋₄ alkoxy-C₁₋₄ alkyl. In some embodiments, R² is methoxy-ethyl.

In some embodiments, R³ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Incertain embodiments, R³ is hydrogen. In some embodiments, R³ is halogen.In certain embodiments, R³ is —NO₂. In some embodiments, R³ is —CN. Incertain embodiments, R³ is —OR. In some embodiments, R³ is C₁₋₁₂aliphatic. In some embodiments, R³ is C₁₋₆ aliphatic.

In some embodiments, R⁴ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Incertain embodiments, R⁴ is hydrogen. In some embodiments, R⁴ is halogen.In certain embodiments, R⁴ is —NO₂. In some embodiments, R⁴ is —CN. Incertain embodiments, R⁴ is —OR. In some embodiments, R⁴ is C₁₋₁₂aliphatic. In some embodiments, R⁴ is C₁₋₆ aliphatic.

In some embodiments, R⁵ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Incertain embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is halogen.In certain embodiments, R⁵ is —NO₂. In some embodiments, R⁵ is —CN. Incertain embodiments, R⁵ is —OR. In some embodiments, R⁵ is C₁₋₁₂aliphatic. In some embodiments, R⁵ is C₁₋₆ aliphatic.

In some embodiments, R⁶ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Incertain embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is halogen.In certain embodiments, R⁶ is —NO₂. In some embodiments, R⁶ is —CN. Incertain embodiments, R⁶ is —OR. In some embodiments, R⁶ is C₁₋₁₂aliphatic. In some embodiments, R⁶ is C₁₋₆ aliphatic.

In some embodiments, R⁷ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Incertain embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is halogen.In certain embodiments, R⁷ is —NO₂. In some embodiments, R⁷ is —CN. Incertain embodiments, R⁷ is —OR. In some embodiments, R⁷ is C₁₋₁₂aliphatic. In some embodiments, R⁷ is C₁₋₆ aliphatic.

In some embodiments, R⁸ is present. In other embodiments, R⁸ is absent.In some embodiments, R⁸ is hydrogen. In some embodiments, R⁸ is halogen.In some embodiments, R⁸ is —OR^(a). In some embodiments, R⁸ isoptionally substituted C₁₋₁₂ aliphatic. In some embodiments, R⁸ is C₁₋₆aliphatic. In some embodiments, R⁸ is C₁₋₃ aliphatic.

In some embodiments, R⁹ is present. In other embodiments, R⁹ is absent.In some embodiments, R⁹ is hydrogen. In some embodiments, R⁹ is halogen.In some embodiments, R⁹ is —OR^(a). In some embodiments, R⁹ isoptionally substituted C₁₋₁₂ aliphatic. In some embodiments, R⁹ is C₁₋₆aliphatic. In some embodiments, R⁹ is C₁₋₃ aliphatic.

In some embodiments, R¹⁰ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Insome embodiments, R¹⁰ is optionally substituted phenyl, a 3-7 memberedsaturated or partially unsaturated monocyclic carbocyclic ring, a 3-7membered saturated or partially unsaturated heterocyclic ring having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur, ora 5-6 membered heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur In certain embodiments, R¹⁰ ishydrogen. In some embodiments, R¹⁰ is halogen. In some embodiments, R¹⁰is C₁₋₁₂ aliphatic. In some embodiments, R¹⁰ is C₁₋₆ aliphatic.

In some embodiments, R¹¹ is hydrogen, halogen, —NO₂, —CN, —OR, —SR,—N(R)₂, —C(O)R, —CO₂R, —C(O)C(O)R, —C(O)CH₂C(O)R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)N(R)₂,—N(R)C(═NR)N(R)₂, —C(═NR)N(R)₂, —C═NOR, —N(R)C(O)N(R)₂, —N(R)SO₂N(R)₂,—N(R)SO₂R, —OC(O)N(R)₂, or optionally substituted C₁₋₁₂ aliphatic. Insome embodiments, R¹¹ is optionally substituted phenyl, a 3-7 memberedsaturated or partially unsaturated monocyclic carbocyclic ring, a 3-7membered saturated or partially unsaturated heterocyclic ring having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur, ora 5-6 membered heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur In certain embodiments, R¹¹ ishydrogen. In some embodiments, R¹¹ is halogen. In some embodiments, R¹¹is C₁₋₁₂ aliphatic. In some embodiments, R¹¹ is C₁₋₆ aliphatic.

It will be appreciated that for compounds depicted herein, wherenegatively charges phosphates are shown, the disclosure contemplatesboth free and salt forms of such compounds, and tautomers thereof. Insome embodiments, a provided compound may have one or more protonatednitrogens that balance the charge of a free phosphate.

In some embodiments, the present invention provides a compound offormula II:

or a pharmaceutically acceptable salt thereof,wherein each of Ring A, Ring B, X^(a), X^(b), X^(c), X^(d), X^(e),X^(f), X^(a1), X^(b1), X², X³, W, R¹, R², R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹is as defined above and described in classes and subclasses herein, bothsingly and in combination.

In some embodiments, a provided compound is other than:

In some embodiments, a provided compound is of formula I-a or II-a:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a provided compound is of formula III, IV, V, VI,VII, VIII, or IX:

or a pharmaceutically acceptable salt thereof, wherein each of Ring A,X^(c), X^(d), X^(e), X^(f), R¹, R², R⁶, R⁷, R⁸, R⁹, X², and X³ is asdefined above and described in classes and subclasses herein, bothsingly and in combination.In some embodiments, a provided compound is of formula X, XI, XII, XIII,XIV, XV, or XVI:

or a pharmaceutically acceptable salt thereof,wherein each of Ring A, X^(c), X^(d), X^(e), X^(f), R¹, R², R⁶, R⁷, R⁸,R⁹, X², and X³ is as defined above and described in classes andsubclasses herein, both singly and in combination.

In some embodiments, a provided compound is selected from:

or a pharmaceutically acceptable salt thereof,wherein each of Ring A, X^(c), X^(d), X^(e), X^(f), R¹, R², R⁶, R⁷, R⁸,R⁹, X², and X³ is as defined above and described in classes andsubclasses herein, both singly and in combination.

In some embodiments, a provided compound is selected from:

or a pharmaceutically acceptable salt thereof,wherein each of Ring A, X^(c), X^(d), X^(e), X^(f), R¹, R², R⁶, R⁷, R⁸,R⁹, X², and X³ is as defined above and described in classes andsubclasses herein, both singly and in combination.

In some embodiments, a provided compound is selected from:

or a pharmaceutically acceptable salt thereof.

It will be appreciated that the compounds depicted in the immediatelypreceding paragraph may be drawn using other conventions. For example,the following two compounds are considered equivalent in chemicalstructure and stereochemistry:

In some embodiments, provided compounds are in isolated form. In someembodiments, provided compounds are pure.

Pharmaceutical Compositions

Provided pharmaceutical compositions can be in a variety of formsincluding oral dosage forms, topic creams, topical patches,iontophoresis forms, suppository, nasal spray and inhaler, eye drops,intraocular injection forms, depot forms, as well as injectable andinfusible solutions. Methods for preparing pharmaceutical compositionare well known in the art.

Pharmaceutical compositions typically contain the active agent describedherein in an amount effective to achieve the desired therapeutic effectwhile avoiding or minimizing adverse side effects. Pharmaceuticallyacceptable preparations and salts of the active agent are providedherein and are well known in the art. For the administration of cGASmodulators and the like, the amount administered desirably is chosenthat is therapeutically effective with few to no adverse side effects.The amount of the therapeutic or pharmaceutical composition which iseffective in the treatment of a particular disease, disorder orcondition depends on the nature and severity of the disease, the targetsite of action, the subject's weight, special diets being followed bythe subject, concurrent medications being used, the administration routeand other factors that are recognized by those skilled in the art. Thedosage can be adapted by the clinician in accordance with conventionalfactors such as the extent of the disease and different parameters fromthe subject. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems (e.g., asdescribed by the U.S. Department of Health and Human Services, Food andDrug Administration, and Center for Drug Evaluation and Research in“Guidance for Industry: Estimating Maximum Safe Starting Dose in InitialClinical Trials for Therapeutics in Adult Healthy Volunteers”,Pharmacology and Toxicology, July 2005, the entire contents of which areincorporated herein by reference).

Various delivery systems are known and can be used to administer activeagent described herein or a pharmaceutical composition comprising thesame.

The pharmaceutical compositions described herein can be administered byany suitable route including, but are not limited to enteral,gastroenteral, epidural, oral, transdermal, epidural (peridural),intracerebral (into the cerebrum), intracerebroventricular (into thecerebral ventricles), epicutaneous (application onto the skin),intradermal, (into the skin itself), subcutaneous (under the skin),nasal administration (through the nose), intravenous (into a vein),intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Other delivery systems well known in the art can be used for delivery ofthe pharmaceutical compositions described herein, for example viaaqueous solutions, encapsulation in microparticules, or microcapsules.The pharmaceutical compositions of the present invention can also bedelivered in a controlled release system. For example, a polymericmaterial can be used (see, e.g., Smolen and Ball, Controlled DrugBioavailability, Drug product design and performance, 1984, John Wiley &Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology andtoxicology series, 2003, 2^(nd) edition, CRRC Press). Alternatively, apump may be used (Saudek et al., N. Engl. J. Med. 321:574 (1989)). Thecompositions described herein may also be coupled to a class ofbiodegradable polymers useful in achieving controlled release of thedrug, for example, polylactic acid, polyorthoesters, cross-linkedamphipathic block copolymers and hydrogels, polyhydroxy butyric acid,and polydihydropyrans.

As described above, pharmaceutical compositions desirably include apharmaceutically acceptable carrier. The term carrier refers todiluents, adjuvants, excipients or vehicles with which modulators areadministered. Such pharmaceutical carriers include sterile liquids suchas water and oils including mineral oil, vegetable oil (e.g., soybeanoil or corn oil), animal oil or oil of synthetic origin. Aqueousglycerol and dextrose solutions as well as saline solutions may also beemployed as liquid carriers of the pharmaceutical compositions of thepresent invention. The choice of the carrier depends on factors wellrecognized in the art, such as the nature of the peptide, peptidederivative or peptidomimetic, its solubility and other physiologicalproperties as well as the target site of delivery and application.Examples of suitable pharmaceutical carriers are described in Remington:The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003,21^(th) edition, Mack Publishing Company. Moreover, suitable carriersfor oral administration are known in the art and are described, forexample, in U.S. Pat. Nos. 6,086,918, 6,673,574, 6,960,355, and7,351,741 and in WO2007/131286, the disclosures of which are herebyincorporated by reference.

Further pharmaceutically suitable materials that may be incorporated inpharmaceutical preparations include absorption enhancers including thoseintended to increase paracellular absorption, pH regulators and buffers,osmolarity adjusters, preservatives, stabilizers, antioxidants,surfactants, thickeners, emollient, dispersing agents, flavoring agents,coloring agents, and wetting agents.

Examples of suitable pharmaceutical excipients include, water, glucose,sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin,starch flour (e.g., rice flour), chalk, sodium stearate, malt, sodiumchloride, and the like. The pharmaceutical compositions comprisingmodulators can take the form of solutions, capsules, tablets, creams,gels, powders sustained release formulations and the like. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides (see Remington: The Science andPractice of Pharmacy by Alfonso R. Gennaro, 2003, 21^(th) edition, MackPublishing Company). Such compositions contain a therapeuticallyeffective amount of the therapeutic composition, together with asuitable amount of carrier so as to provide the form for properadministration to the subject. The formulations are designed to suit themode of administration and the target site of action (e.g., a particularorgan or cell type).

The pharmaceutical compositions comprising the active agent describedherein also include compositions formulated as neutral or salt forms.Pharmaceutically acceptable salts include those that form with freeamino groups and those that react with free carboxyl groups. Non-toxicalkali metal, alkaline earth metal, and ammonium salts commonly used inthe pharmaceutical industry include sodium, potassium, lithium, calcium,magnesium, barium, ammonium, and protamine zinc salts, which areprepared by methods well known in the art. Also included are non-toxicacid addition salts, which are generally prepared by reacting thecompounds of the present invention with suitable organic or inorganicacid. Representative salts include the hydrobromide, hydrochloride,valerate, oxalate, oleate, laureate, borate, benzoate, sulfate,bisulfate, acetate, phosphate, tysolate, citrate, maleate, fumarate,tartrate, succinate, napsylate salts, and the like.

Examples of fillers or binders that may be used in accordance with thepresent invention include acacia, alginic acid, calcium phosphate(dibasic), carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, dextrin, dextrates, sucrose, tylose,pregelatinized starch, calcium sulfate, amylose, glycine, bentonite,maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodiumphosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose,guar gum, liquid glucose, compressible sugar, magnesium aluminumsilicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone,sodium alginate, tragacanth microcrystalline cellulose, starch, andzein. In certain embodiments, a filler or binder is microcrystallinecellulose.

Examples of disintegrating agents that may be used include alginic acid,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxypropylcellulose (low substituted), microcrystalline cellulose,powdered cellulose, colloidal silicon dioxide, sodium croscarmellose,crospovidone, methylcellulose, polacrilin potassium, povidone, sodiumalginate, sodium starch glycolate, starch, disodium disulfite, disodiumedathamil, disodium edetate, disodiumethylenediaminetetraacetate (EDTA)crosslinked polyvinylpyrrolidones, pregelatinized starch, carboxymethylstarch, sodium carboxymethyl starch, microcrystalline cellulose.

Examples of lubricants include calcium stearate, canola oil, glycerylpalmitostearate, hydrogenated vegetable oil (type I), magnesium oxide,magnesium stearate, mineral oil, poloxamer, polyethylene glycol, sodiumlauryl sulfate, sodium stearate fumarate, stearic acid, talc and, zincstearate, glyceryl behapate, magnesium lauryl sulfate, boric acid,sodium benzoate, sodium acetate, sodium benzoate/sodium acetate (incombination), DL-leucine.

Examples of silica flow conditioners include colloidal silicon dioxide,magnesium aluminum silicate and guar gum. Another most preferred silicaflow conditioner consists of silicon dioxide.

Examples of stabilizing agents include acacia, albumin, polyvinylalcohol, alginic acid, bentonite, dicalcium phosphate,carboxymethylcellulose, hydroxypropylcellulose, colloidal silicondioxide, cyclodextrins, glyceryl monostearate, hydroxypropylmethylcellulose, magnesium trisilicate, magnesium aluminum silicate,propylene glycol, propylene glycol alginate, sodium alginate, carnaubawax, xanthan gum, starch, stearate(s), stearic acid, stearicmonoglyceride and stearyl alcohol.

In some embodiments, the present invention contemplates oralformulations containing the active agent described herein. For example,pharmaceutical compositions described herein may include a cyclodextrinor cyclodextrin derivative. Cyclodextrins are generally made up of fiveor more α-D-glycopyranoside unites linked 1→4. Typically, cyclodextrinscontain a number of glucose monomers ranging from six to eight units ina ring, creating a cone shape (α-cyclodextrin: six membered sugar ringmolecule, β-cyclodextrin: seven sugar ring molecule, γ-cyclodextrin:eight sugar ring molecule). Exemplary cyclodextrins and cyclodextrinderivatives are disclosed in U.S. Pat. No. 7,723,304, U.S. PublicationNo. 2010/0196452, and U.S. Publication No. 2010/0144624, the entirecontents of each of which are incorporated herein by reference. Forexample, in some embodiments, a cyclodextrin in accordance with thepresent invention is an alkylated cyclodextrin, hydroxyalkylatedcyclodextrin, or acylated cyclodextrin. In some embodiments, acyclodextrin is a hydroxypropyl β-cyclodextrin. Exemplary cyclodextrinderivatives are disclosed in Szejtli, J. Chem Rev, (1998), 98,1743-1753; and Szente, L and Szejtli, J., Advance Drug Delivery Reviews,36 (1999) 17-28, the entire contents of each of which are herebyincorporated by reference. Examples of cyclodextin derivatives includemethylated cyclodextrins (e.g., RAMEB; randomly methylatedβ-cyclodextrin); hydroxyalkylated cyclodextrins(hydroxypropyl-β-cyclodextrin and hydroxypropyl γ-cyclodextrin);acetylated cyclodextrins (acetyl-γ-cyclodextrin); reactive cyclodextrins(chlorotriazinyl β-cyclodextrin); and branched cyclodextrins (glucosyl-and maltosyl β-cyclodextrin); acetyl-γ-cyclodextrin;acetyl-β-cyclodextrin, sulfobutyl-β cyclodextrin, sulfated α-, β- andγ-cyclodextrins; sulfoalkylated cyclodextrins; and hydroxypropylβ-cyclodextrin.

Dosing

Typically, active agent described herein in an amount ranging from 0.001to 100 mg/kg/day is administered to the subject. For example, in someembodiments, about 0.01 mg/kg/day to about 25 mg/kg/day, about 1mg/kg/day to about 20 mg/kg/day, 0.2 mg/kg/day to about 10 mg/kg/day,about 0.02 mg/kg/day to about 0.1 mg/kg/day, or about 1 mg/kg/day toabout 100 mg/kg/day is administered to the subject. In some embodiments,active agent described herein in an amount of about 10 μg/kg/day, 50μg/kg/day, 100 μg/kg/day, 200 μg/kg/day, 300 μg/kg/day, 400 μg/kg/day,500 μg/kg/day, 600 μg/kg/day, 700 μg/kg/day, 800 μg/kg/day, 900μg/kg/day, or 1000 μg/kg/day is administered to the subject.

In some embodiments, the compound is administered at an effective doseranging from about 1-1,000 μg/kg/day (e.g., ranging from about 1-900μg/kg/day, 1-800 μg/kg/day, 1-700 μg/kg/day, 1-600 μg/kg/day, 1-500μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200 μg/kg/day, 1-100μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70 μg/kg/day, 1-60μg/kg/day, 1-50 μg/kg/day, 1-40 μg/kg/day, 1-30 μg/kg/day, 1-20μg/kg/day, 1-10 μg/kg/day). In some embodiments, the compound isadministered at an effective dose ranging from about 1-500 μg/kg/day. Insome embodiments, the compound is administered at an effective doseranging from about 1-100 μg/kg/day. In some embodiments, the compound isadministered at an effective dose ranging from about 1-60 μg/kg/day. Insome embodiments, the compound is administered at an effective doseselected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1,000 μg/kg/day.

In some embodiments, a therapeutically effective amount of a compoundmay be an amount ranging from about 10-1,000 mg (e.g., about 20 mg-1,000mg, 30 mg-1,000 mg, 40 mg-1,000 mg, 50 mg-1,000 mg, 60 mg-1,000 mg, 70mg-1,000 mg, 80 mg-1,000 mg, 90 mg-1,000 mg, about 10-900 mg, 10-800 mg,10-700 mg, 10-600 mg, 10-500 mg, 100-1000 mg, 100-900 mg, 100-800 mg,100-700 mg, 100-600 mg, 100-500 mg, 100-400 mg, 100-300 mg, 200-1000 mg,200-900 mg, 200-800 mg, 200-700 mg, 200-600 mg, 200-500 mg, 200-400 mg,300-1000 mg, 300-900 mg, 300-800 mg, 300-700 mg, 300-600 mg, 300-500 mg,400 mg-1,000 mg, 500 mg-1,000 mg, 100 mg-900 mg, 200 mg-800 mg, 300mg-700 mg, 400 mg-700 mg, and 500 mg-600 mg). In some embodiments, acompound is present in an amount of or greater than about 10 mg, 50 mg,100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg,550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg. In some embodiments, acompound is present in an amount of or less than about 1000 mg, 950 mg,900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg,450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100 mg. Insome embodiments, the therapeutically effective amount described hereinis provided in one dose. In some embodiments, the therapeuticallyeffective amount described herein is provided in one day.

In other embodiments, a therapeutically effective amount may be, forexample, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., from about0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kg weight to300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kg weight,from about 0.001 mg/kg weight to 100 mg/kg weight, from about 0.001mg/kg weight to 90 mg/kg weight, from about 0.001 mg/kg weight to 80mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, fromabout 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kgweight to 50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kgweight, from about 0.001 mg/kg weight to 30 mg/kg weight, from about0.001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to20 mg/kg weight, from about 0.001 mg/kg weight to 15 mg/kg weight, fromabout 0.001 mg/kg weight to 10 mg/kg weight. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective amount may be,for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g. fromabout 0.0001 mg/kg weight to 0.09 mg/kg weight, from about 0.0001 mg/kgweight to 0.08 mg/kg weight, from about 0.0001 mg/kg weight to 0.07mg/kg weight, from about 0.0001 mg/kg weight to 0.06 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.05 mg/kg weight, from about 0.0001 mg/kgweight to about 0.04 mg/kg weight, from about 0.0001 mg/kg weight to0.03 mg/kg weight, from about 0.0001 mg/kg weight to 0.02 mg/kg weight,from about 0.0001 mg/kg weight to 0.019 mg/kg weight, from about 0.0001mg/kg weight to 0.018 mg/kg weight, from about 0.0001 mg/kg weight to0.017 mg/kg weight, from about 0.0001 mg/kg weight to 0.016 mg/kgweight, from about 0.0001 mg/kg weight to 0.015 mg/kg weight, from about0.0001 mg/kg weight to 0.014 mg/kg weight, from about 0.0001 mg/kgweight to 0.013 mg/kg weight, from about 0.0001 mg/kg weight to 0.012mg/kg weight, from about 0.0001 mg/kg weight to 0.011 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.01 mg/kg weight, from about 0.0001 mg/kgweight to 0.009 mg/kg weight, from about 0.0001 mg/kg weight to 0.008mg/kg weight, from about 0.0001 mg/kg weight to 0.007 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.006 mg/kg weight, from about 0.0001 mg/kgweight to 0.005 mg/kg weight, from about 0.0001 mg/kg weight to 0.004mg/kg weight, from about 0.0001 mg/kg weight to 0.003 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.002 mg/kg weight. In some embodiments,the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002mg/kg weight, 0.0003 mg/kg weight, 0.0004 mg/kg weight, 0.0005 mg/kgweight, 0.0006 mg/kg weight, 0.0007 mg/kg weight, 0.0008 mg/kg weight,0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kgweight, 0.004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight,0.007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kgweight, 0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05mg/kg weight, 0.06 mg/kg weight, 0.07 mg/kg weight, 0.08 mg/kg weight,0.09 mg/kg weight, or 0.1 mg/kg weight. The effective dose for aparticular individual can be varied (e.g., increased or decreased) overtime, depending on the needs of the individual. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In some embodiments, a formulation comprising a compound as describedherein administered as a single dose. In some embodiments, a formulationcomprising a compound as described herein is administered at regularintervals. Administration at an “interval,” as used herein, indicatesthat the therapeutically effective amount is administered periodically(as distinguished from a one-time dose). The interval can be determinedby standard clinical techniques. In some embodiments, a formulationcomprising a compound as described herein is administered bimonthly,monthly, twice monthly, triweekly, biweekly, weekly, twice weekly,thrice weekly, daily, twice daily, or every six hours. Theadministration interval for a single individual need not be a fixedinterval, but can be varied over time, depending on the needs of theindividual.

As used herein, the term “bimonthly” means administration once per twomonths (i.e., once every two months); the term “monthly” meansadministration once per month; the term “triweekly” means administrationonce per three weeks (i.e., once every three weeks); the term “biweekly”means administration once per two weeks (i.e., once every two weeks);the term “weekly” means administration once per week; and the term“daily” means administration once per day.

In some embodiments, a formulation comprising a compound as describedherein is administered at regular intervals indefinitely. In someembodiments, a formulation comprising a compound as described herein isadministered at regular intervals for a defined period. In someembodiments, a formulation comprising a compound as described herein isadministered at regular intervals for 5 years, 4, years, 3, years, 2,years, 1 year, 11 months, 10 months, 9 months, 8 months, 7 months, 6months, 5 months, 4 months, 3 months, 2 months, a month, 3 weeks, 2,weeks, a week, 6 days, 5 days, 4 days, 3 days, 2 days, or a day.

Methods of Use

In certain embodiments provided compounds are useful in medicine. Insome embodiments, provided compounds are useful in treating immunedisease, disorders, or conditions. In some embodiments, the presentinvention provides a method for the treatment or prevention of an immunedisease, disorder, or condition comprising administering to a subject inneed thereof a provided compound or a pharmaceutical compositionthereof.

In some embodiments, the immune disease, disorder, or condition is anautoimmune disease, disorder, or condition. In certain embodiments, theimmune disease, disorder, or condition is selected from the groupconsisting of any of a variety of diseases, disorders, and/orconditions, including but not limited to one or more of the following:autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis,psoriasis, rheumatoid arthritis); inflammatory disorders (e.g.arthritis, pelvic inflammatory disease); infectious diseases (e.g. viralinfections (e.g., HIV, HCV, RSV), bacterial infections, fungalinfections, sepsis); neurological disorders (e g. Alzheimer's disease,Huntington's disease; autism; Duchenne muscular dystrophy);cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia,thrombosis, clotting disorders, angiogenic disorders such as maculardegeneration); proliferative disorders (e.g. cancer, benign neoplasms);respiratory disorders (e.g. chronic obstructive pulmonary disease);digestive disorders (e.g. inflammatory bowel disease, ulcers);musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine,metabolic, and nutritional disorders (e.g. diabetes, osteoporosis);urological disorders (e.g. renal disease); psychological disorders (e.g.depression, schizophrenia); skin disorders (e.g. wounds, eczema); bloodand lymphatic disorders (e.g. anemia, hemophilia); etc. In someembodiments, the immune disease, disorder, or condition is characterizedby inflammation. In some embodiments, the immune disease, disorder, orcondition is caused by, sustained by, or related to cGAS activation. Insome embodiments, the immune disease, disorder, or condition is causedby, sustained by, or related to STING activation.

In some embodiments the autoimmune disorder or disease is selected fromAcute disseminated encephalomyelitis (ADEM), Addison's disease,Agammaglobulinemia, Alopecia areata, Amyotrophic lateral sclerosis (AlsoLou Gehrig's disease; Motor Neuron Disease), Ankylosing Spondylitis,Antiphospholipid syndrome, Antisynthetase syndrome, Atopic allergy,Atopic dermatitis, Autoimmune aplastic anemia, Autoimmunecardiomyopathy, Autoimmune enteropathy, Autoimmune hemolytic anemia,Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmunelymphoproliferative syndrome, Autoimmune peripheral neuropathy,Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmuneprogesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmuneurticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis,Behçet's disease, Berger's disease, Bickerstaffs encephalitis, Blausyndrome, Bullous pemphigoid Cancer, Castleman's disease, Celiacdisease, Chagas disease, Chronic inflammatory demyelinatingpolyneuropathy, Chronic recurrent multifocal osteomyelitis, Chronicobstructive pulmonary disease, Churg-Strauss syndrome, Cicatricialpemphigoid Cogan syndrome, Cold agglutinin disease, Complement component2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome,Crohn's disease (idiopathic inflammatory bowel disease “IBD”), Cushing'sSyndrome, Cutaneous leukocytoclastic angiitis, Dego's disease,Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1,Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Drug-inducedlupus, Discoid lupus erythematosus, Eczema, Endometriosis,Enthesitis-related arthritis, Eosinophilic fasciitis, Eosinophilicgastroenteritis, Epidermolysis bullosa acquisita, Erythema nodosum,Erythroblastosis fetalis, Essential mixed cryoglobulinemia, Evan'ssyndrome, Fibrodysplasia ossificans progressiva, Fibrosing alveolitis(or Idiopathic pulmonary fibrosis), Gastritis, Gastrointestinalpemphigoid, Glomerulonephritis, Goodpasture's syndrome, Graves' disease,Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto'sthyroiditis, Henoch-Schonlein purpura, Herpes gestationis akaGestational Pemphigoid, Hidradenitis suppurativa, Hughes-Stovinsyndrome, Hypogammaglobulinemia, Idiopathic inflammatory demyelinatingdiseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenicpurpura, IgA nephropathy, Inclusion body myositis, Chronic inflammatorydemyelinating polyneuropathy, Interstitial cystitis, Juvenile idiopathicarthritis aka Juvenile rheumatoid arthritis, Kawasaki's disease,Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichenplanus, Lichen sclerosus, Linear IgA disease (LAD), Lupoid hepatitis akaAutoimmune hepatitis, Lupus erythematosus, Majeed syndrome, Ménière'sdisease, Microscopic polyangiitis, Miller-Fisher syndrome seeGuillain-Barre Syndrome, Mixed connective tissue disease, Morphea,Mucha-Habermann disease aka Pityriasis lichenoides et varioliformisacuta, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (also Devic's disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord'sthyroiditis, Palindromic rheumatism, PANDAS (pediatric autoimmuneneuropsychiatric disorders associated with streptococcus),Paraneoplastic cerebellar degeneration, Paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome,Pars planitis, Pemphigus vulgaris, Pernicious anaemia, Perivenousencephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgiarheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosingcholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriaticarthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen'sencephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter'ssyndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoidarthritis, Rheumatic fever, Sarcoidosis, Schizophrenia, Schmidtsyndrome, Schnitzler syndrome, Scleritis, Scleroderma, Serum Sickness,Sjögren's syndrome, Spondyloarthropathy, Still's disease, Subacutebacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome,Sydenham chorea see PANDAS, Sympathetic ophthalmia, Systemic lupuserythematosis, Takayasu's arteritis, Temporal arteritis (also known as“giant cell arteritis”), Thrombocytopenia, Tolosa-Hunt syndrome,Transverse myelitis, Ulcerative colitis (one of two types of idiopathicinflammatory bowel disease “IBD”), Undifferentiated connective tissuedisease different from Mixed connective tissue disease, Undifferentiatedspondyloarthropathy, Urticarial vasculitis, Vasculitis, Vitiligo, andWegener's granulomatosis.

In certain embodiments, administration of a compound to a patient inneed thereof results in a decrease of cGAS activity. In someembodiments, administration of a compound to a patient in need thereofresults in a decrease of STING activity.

In some embodiments, compounds used in the provided methods are preparedby chemical synthesis.

In certain embodiments, the present invention provides a method ofinhibiting cGAS comprising contacting cGAS with a provided compound. Insome embodiments, the present invention provides a method of inhibitingcGAS in a patient comprising administering to a patient a providedcompound. In certain embodiments, the present invention provides amethod of inhibiting STING comprising contacting STING with a providedcompound. In some embodiments, the present invention provides a methodof inhibiting STING in a patient comprising administering to a patient aprovided compound.

In certain embodiments, the present invention provides a method ofmodulating activity of an cGAS polypeptide, the method comprisingcontacting the cGAS polypeptide with a cGAS modulator designed by themethods disclosed herein, which modulating agent is not a knownmodulator, substrate, or product of cGAS. In some embodiments, themodulating agent is a provided compound.

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (compounds and compositions as described above) of theinvention. In one embodiment, the kit comprises one or more functionalantibodies or function fragments thereof.

Kits of the invention may comprise one or more cGAMP parent molecules,or any mimic, analog or variant thereof. Kits may also comprise any ofthe cGAS variants, derivatives or mutants described herein. The kit mayfurther comprise packaging and instructions and/or a delivery agent toform a formulation composition. The delivery agent may comprise asaline, a buffered solution, a lipid or any delivery agent disclosedherein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions may be precipitated or it maybe lyophilized. The amount of each component may be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components may also be varied in order to increase thestability of the compound or composition in the buffer solution over aperiod of time and/or under a variety of conditions. In one aspect, thepresent invention provides kits for research applications related tocGAS activity or cGAMP signaling, provided in an amount effective tostudy the concomitant signaling pathways when introduced into a targetcell. The kits may further comprise a second or further compound orcomposition described herein. Such second or further molecules maymodulate the immune response or an inflammatory process or comprise oneor more therapeutic molecules. In one embodiment, a kit comprises atleast one cGAS polypeptide and at least one cGAMP molecule. In oneembodiment, the kits of the present invention comprise packaging andinstructions.

cGAS Crystal Structures

Among other things, the present invention provides a crystalline (i.e.,containing at least one crystal) or crystallizable compositioncomprising an cGAS polypeptide as described herein (see also Gao et al.Cell 153, 1094-1107 (2013), including supplementary materials). In someembodiments, such a provided composition consists of or consistsessentially of the cGAS polypeptide. In some embodiments, a compositionis considered to “consist of” cGAS polypeptide if it includes only thepolypeptide, one or more solvents, and optionally salts and/or metals.In some embodiments, such a provided composition includes one or moreother agents such as one or more other polypeptides (e.g., one or morepotential or actual cGAS binding partner polypeptides or nucleic acids)and/or one or more interacting agents (e.g., small molecules).

The present invention also provides structural information and/oranalyses of cGAS polypeptide crystals and/or sets thereof. In someembodiments, such structural information includes, but is not limitedto, diffraction patterns, and/or coordinates, as well as any data sets,images, models, and/or graphical representations thereof or generatedtherefrom. In some embodiments, such graphical representations mayinclude, for example, space-filling models, molecular surfacerepresentations, shell or boundary models, ribbon models, stick models;and/or combinations thereof.

In some embodiments, provided information is or comprises differencesobserved between or among structures that differ from one another in thepresence or absence of one or more binding partners and/or interactingagents. In some embodiments, provided information is or comprisesdifferences observed between or among structures that differ from oneanother in the presence or absence of one or more binding partnersand/or one or more modulators.

In some embodiments, such structural information and/or analyses may beembodied in a tangible medium (e.g., a computer-readable medium) or astorage environment. Thus, the present invention provides tangibleembodiments of cGAS polypeptide crystal structure information, as wellas its use, for example, by or with a computer system, in any of avariety of applications. For example, in some embodiments, suchstructural information and/or analyses may be accessed by, transportedto or from, and/or otherwise utilized by a computer system or programrunning thereon.

Structure-Based Drug Design

In some embodiments, the present disclosure provides systems foridentifying and/or characterizing cGAS modulators. In some embodiments,the present disclosure provides a method of designing or characterizinga cGAS modulator comprising the steps of:

a) providing an image of a cGAS crystal that includes at least onepotential interaction site;

b) docking in the image at least one moiety that is a potential cGASmodulator structural element; and

c) assessing one or more features of a potential moiety-interaction siteinteraction.

In some embodiments, the at least one potential interaction siteincludes a site selected from the group consisting of Ser199, Ser420,Lys402, Glu211, Asp213, Asp307, Tyr421, Arg364, and combinationsthereof. In certain embodiments, the at least one potential interactionsite includes a site selected from the group consisting of Tyr421,Thr197, Ser366, Ser368, Arg364, and combinations thereof. In certainembodiments, the at least one potential interaction site includes a siteselected from the group consisting of Tyr421, Asp213, Asp307, Arg364,and combinations thereof. In some embodiments, the at least onepotential interaction site includes Arg161. In some embodiments, themodulator is a compound disclosed herein.

In some embodiments, the one or more features include at least onefeature selected from the group consisting of: spatial separationbetween the moiety and the potential interaction site; energy of thepotential moiety-interaction site interaction, and/or combinationsthereof.

In some embodiments, a method further comprises a step of providing animage of a potential cGAS modulator comprising the moiety docked withthe image of the cGAS crystal. In some embodiments, a method furthercomprises a step of comparing the image with that of a cGAS crystalincluding a bound known modulator, substrate, or product.

Computer Systems

As will be appreciated by those skilled in the art, reading the presentdisclosure, in some aspects, the present invention is ideally suited foruse in computer-implemented inventions. As shown in FIG. 17, animplementation of an exemplary cloud computing environment 2400 is shownand described. The cloud computing environment 2400 may include one ormore resource providers 2402 a, 2402 b, 2402 c (collectively, 2402).Each resource provider 2402 may include computing resources. In someimplementations, computing resources may include any hardware and/orsoftware used to process data. For example, computing resources mayinclude hardware and/or software capable of executing algorithms,computer programs, and/or computer applications. In someimplementations, exemplary computing resources may include applicationservers and/or databases with storage and retrieval capabilities. Eachresource provider 2402 may be connected to any other resource provider2402 in the cloud computing environment 2400. In some implementations,the resource providers 2402 may be connected over a computer network2408. Each resource provider 2402 may be connected to one or morecomputing device 2404 a, 2404 b, 2404 c (collectively, 2404), over thecomputer network 2408.

The cloud computing environment 2400 may include a resource manager2406. The resource manager 2406 may be connected to the resourceproviders 2402 and the computing devices 2404 over the computer network2408. In some implementations, the resource manager 2406 may facilitatethe provision of computing resources by one or more resource providers2402 to one or more computing devices 2404. The resource manager 2406may receive a request for a computing resource from a particularcomputing device 2404. The resource manager 2406 may identify one ormore resource providers 2402 capable of providing the computing resourcerequested by the computing device 2404. The resource manager 2406 mayselect a resource provider 2402 to provide the computing resource. Theresource manager 2406 may facilitate a connection between the resourceprovider 2402 and a particular computing device 2404. In someimplementations, the resource manager 2406 may establish a connectionbetween a particular resource provider 2402 and a particular computingdevice 2404. In some implementations, the resource manager 2406 mayredirect a particular computing device 2404 to a particular resourceprovider 2402 with the requested computing resource.

FIG. 18 shows an example of a computing device 2500 and a mobilecomputing device 2550 that can be used to implement the techniquesdescribed in this disclosure. The computing device 2500 is intended torepresent various forms of digital computers, such as laptops, desktops,workstations, personal digital assistants, servers, blade servers,mainframes, and other appropriate computers. The mobile computing device2550 is intended to represent various forms of mobile devices, such aspersonal digital assistants, cellular telephones, smart-phones, tabletcomputers, and other similar computing devices. The components shownhere, their connections and relationships, and their functions, aremeant to be examples only, and are not meant to be limiting.

The computing device 2500 includes a processor 2502, a memory 2504, astorage device 2506, a high-speed interface 2508 connecting to thememory 2504 and multiple high-speed expansion ports 2510, and alow-speed interface 2512 connecting to a low-speed expansion port 2514and the storage device 2506. Each of the processor 2502, the memory2504, the storage device 2506, the high-speed interface 2508, thehigh-speed expansion ports 2510, and the low-speed interface 2512, areinterconnected using various busses, and may be mounted on a commonmotherboard or in other manners as appropriate. The processor 2502 canprocess instructions for execution within the computing device 2500,including instructions stored in the memory 2504 or on the storagedevice 2506 to display graphical information for a GUI on an externalinput/output device, such as a display 2516 coupled to the high-speedinterface 2508. In other implementations, multiple processors and/ormultiple buses may be used, as appropriate, along with multiple memoriesand types of memory. Also, multiple computing devices may be connected,with each device providing portions of the necessary operations (e.g.,as a server bank, a group of blade servers, or a multi-processorsystem).

The memory 2504 stores information within the computing device 2500. Insome implementations, the memory 2504 is a volatile memory unit orunits. In some implementations, the memory 2504 is a non-volatile memoryunit or units. The memory 2504 may also be another form ofcomputer-readable medium, such as a magnetic or optical disk.

The storage device 2506 is capable of providing mass storage for thecomputing device 2500. In some implementations, the storage device 2506may be or contain a computer-readable medium, such as a floppy diskdevice, a hard disk device, an optical disk device, or a tape device, aflash memory or other similar solid state memory device, or an array ofdevices, including devices in a storage area network or otherconfigurations. Instructions can be stored in an information carrier.The instructions, when executed by one or more processing devices (forexample, processor 2502), perform one or more methods, such as thosedescribed above. The instructions can also be stored by one or morestorage devices such as computer- or machine-readable mediums (forexample, the memory 2504, the storage device 2506, or memory on theprocessor 2502).

The high-speed interface 2508 manages bandwidth-intensive operations forthe computing device 2500, while the low-speed interface 2512 manageslower bandwidth-intensive operations. Such allocation of functions is anexample only. In some implementations, the high-speed interface 2508 iscoupled to the memory 2504, the display 2516 (e.g., through a graphicsprocessor or accelerator), and to the high-speed expansion ports 2510,which may accept various expansion cards (not shown). In theimplementation, the low-speed interface 2512 is coupled to the storagedevice 2506 and the low-speed expansion port 2514. The low-speedexpansion port 2514, which may include various communication ports(e.g., USB, Bluetooth®, Ethernet, wireless Ethernet) may be coupled toone or more input/output devices, such as a keyboard, a pointing device,a scanner, or a networking device such as a switch or router, e.g.,through a network adapter.

The computing device 2500 may be implemented in a number of differentforms, as shown in the figure. For example, it may be implemented as astandard server 2520, or multiple times in a group of such servers. Inaddition, it may be implemented in a personal computer such as a laptopcomputer 2522. It may also be implemented as part of a rack serversystem 2524. Alternatively, components from the computing device 2500may be combined with other components in a mobile device (not shown),such as a mobile computing device 2550. Each of such devices may containone or more of the computing device 2500 and the mobile computing device2550, and an entire system may be made up of multiple computing devicescommunicating with each other.

The mobile computing device 2550 includes a processor 2552, a memory2564, an input/output device such as a display 2554, a communicationinterface 2566, and a transceiver 2568, among other components. Themobile computing device 2550 may also be provided with a storage device,such as a micro-drive or other device, to provide additional storage.Each of the processor 2552, the memory 2564, the display 2554, thecommunication interface 2566, and the transceiver 2568, areinterconnected using various buses, and several of the components may bemounted on a common motherboard or in other manners as appropriate.

The processor 2552 can execute instructions within the mobile computingdevice 2550, including instructions stored in the memory 2564. Theprocessor 2552 may be implemented as a chipset of chips that includeseparate and multiple analog and digital processors. The processor 2552may provide, for example, for coordination of the other components ofthe mobile computing device 2550, such as control of user interfaces,applications run by the mobile computing device 2550, and wirelesscommunication by the mobile computing device 2550.

The processor 2552 may communicate with a user through a controlinterface 2558 and a display interface 2556 coupled to the display 2554.The display 2554 may be, for example, a TFT (Thin-Film-Transistor LiquidCrystal Display) display or an OLED (Organic Light Emitting Diode)display, or other appropriate display technology. The display interface2556 may comprise appropriate circuitry for driving the display 2554 topresent graphical and other information to a user. The control interface2558 may receive commands from a user and convert them for submission tothe processor 2552. In addition, an external interface 2562 may providecommunication with the processor 2552, so as to enable near areacommunication of the mobile computing device 2550 with other devices.The external interface 2562 may provide, for example, for wiredcommunication in some implementations, or for wireless communication inother implementations, and multiple interfaces may also be used.

The memory 2564 stores information within the mobile computing device2550. The memory 2564 can be implemented as one or more of acomputer-readable medium or media, a volatile memory unit or units, or anon-volatile memory unit or units. An expansion memory 2574 may also beprovided and connected to the mobile computing device 2550 through anexpansion interface 2572, which may include, for example, a SIMM (SingleIn Line Memory Module) card interface. The expansion memory 2574 mayprovide extra storage space for the mobile computing device 2550, or mayalso store applications or other information for the mobile computingdevice 2550. Specifically, the expansion memory 2574 may includeinstructions to carry out or supplement the processes described above,and may include secure information also. Thus, for example, theexpansion memory 2574 may be provide as a security module for the mobilecomputing device 2550, and may be programmed with instructions thatpermit secure use of the mobile computing device 2550. In addition,secure applications may be provided via the SIMM cards, along withadditional information, such as placing identifying information on theSIMM card in a non-hackable manner.

The memory may include, for example, flash memory and/or NVRAM memory(non-volatile random access memory), as discussed below. In someimplementations, instructions are stored in an information carrier. thatthe instructions, when executed by one or more processing devices (forexample, processor 2552), perform one or more methods, such as thosedescribed above. The instructions can also be stored by one or morestorage devices, such as one or more computer- or machine-readablemediums (for example, the memory 2564, the expansion memory 2574, ormemory on the processor 2552). In some implementations, the instructionscan be received in a propagated signal, for example, over thetransceiver 2568 or the external interface 2562.

The mobile computing device 2550 may communicate wirelessly through thecommunication interface 2566, which may include digital signalprocessing circuitry where necessary. The communication interface 2566may provide for communications under various modes or protocols, such asGSM voice calls (Global System for Mobile communications), SMS (ShortMessage Service), EMS (Enhanced Messaging Service), or MMS messaging(Multimedia Messaging Service), CDMA (code division multiple access),TDMA (time division multiple access), PDC (Personal Digital Cellular),WCDMA (Wideband Code Division Multiple Access), CDMA2000, or GPRS(General Packet Radio Service), among others. Such communication mayoccur, for example, through the transceiver 2568 using aradio-frequency. In addition, short-range communication may occur, suchas using a Bluetooth®, Wi-Fi™, or other such transceiver (not shown). Inaddition, a GPS (Global Positioning System) receiver module 2570 mayprovide additional navigation- and location-related wireless data to themobile computing device 2550, which may be used as appropriate byapplications running on the mobile computing device 2550.

The mobile computing device 2550 may also communicate audibly using anaudio codec 2560, which may receive spoken information from a user andconvert it to usable digital information. The audio codec 2560 maylikewise generate audible sound for a user, such as through a speaker,e.g., in a handset of the mobile computing device 2550. Such sound mayinclude sound from voice telephone calls, may include recorded sound(e.g., voice messages, music files, etc.) and may also include soundgenerated by applications operating on the mobile computing device 2550.

The mobile computing device 2550 may be implemented in a number ofdifferent forms, as shown in the figure. For example, it may beimplemented as a cellular telephone 2580. It may also be implemented aspart of a smart-phone 2582, personal digital assistant, or other similarmobile device.

Various implementations of the systems and techniques described here canbe realized in digital electronic circuitry, integrated circuitry,specially designed ASICs (application specific integrated circuits),computer hardware, firmware, software, and/or combinations thereof.These various implementations can include implementation in one or morecomputer programs that are executable and/or interpretable on aprogrammable system including at least one programmable processor, whichmay be special or general purpose, coupled to receive data andinstructions from, and to transmit data and instructions to, a storagesystem, at least one input device, and at least one output device.

These computer programs (also known as programs, software, softwareapplications or code) include machine instructions for a programmableprocessor, and can be implemented in a high-level procedural and/orobject-oriented programming language, and/or in assembly/machinelanguage. As used herein, the terms machine-readable medium andcomputer-readable medium refer to any computer program product,apparatus and/or device (e.g., magnetic discs, optical disks, memory,Programmable Logic Devices (PLDs)) used to provide machine instructionsand/or data to a programmable processor, including a machine-readablemedium that receives machine instructions as a machine-readable signal.The term machine-readable signal refers to any signal used to providemachine instructions and/or data to a programmable processor.

To provide for interaction with a user, the systems and techniquesdescribed here can be implemented on a computer having a display device(e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor)for displaying information to the user and a keyboard and a pointingdevice (e.g., a mouse or a trackball) by which the user can provideinput to the computer. Other kinds of devices can be used to provide forinteraction with a user as well; for example, feedback provided to theuser can be any form of sensory feedback (e.g., visual feedback,auditory feedback, or tactile feedback); and input from the user can bereceived in any form, including acoustic, speech, or tactile input.

The systems and techniques described here can be implemented in acomputing system that includes a back end component (e.g., as a dataserver), or that includes a middleware component (e.g., an applicationserver), or that includes a front end component (e.g., a client computerhaving a graphical user interface or a Web browser through which a usercan interact with an implementation of the systems and techniquesdescribed here), or any combination of such back end, middleware, orfront end components. The components of the system can be interconnectedby any form or medium of digital data communication (e.g., acommunication network). Examples of communication networks include alocal area network (LAN), a wide area network (WAN), and the Internet.

The computing system can include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other.

In certain embodiments, the present invention provides a systemcomprising a computer or computer readable medium in which a cGAScrystal structure, or coordinates thereof, is embedded and/or displayed.

In some embodiments, the present invention provides a method ofdesigning and/or characterizing an cGAS modulator, which methodcomprises steps of:

(i) using a provided system to assess one or more structural features ofthe cGAS modulator; and

(ii) performing one or more in vitro, in vivo or cell-based assays tocharacterize the cGAS modulator.

In some embodiments, the method further comprises the step of performinga competition experiment between the cGAS modulator and a known cGASmodulator, substrate, or product. In some embodiments, the methodfurther comprises the step of defining the three-dimensional shape ofthe inhibitor.

In some embodiments, the present invention provides a computer systemcontaining a set of information to perform a design or characterizationof an cGAS inhibitor having a user interface comprising a display unit,the set of information comprising:

(i) logic for inputting an information regarding a binding of a cGASprotein to a moiety known to bind cGAS protein;

(ii) logic for design a candidate cGAS inhibitor based on the binding ofthe cGAS protein to the moiety known to bind cGAS protein;

(iii) logic for determining an information regarding a binding of thecGAS protein to the candidate cGAS inhibitor; and

(iv) logic for making a conclusion regarding a cGAS inhibitoryproperties of the candidate cGAS inhibitor based on the determination ofstep (iii).

In some embodiments, the present invention provides a computer-readablestorage medium containing a set of information for a general purposecomputer having a user interface comprising, a display unit, the set ofinformation comprising:

(i) logic for inputting an information regarding a binding of a cGASprotein to a chemical known to binding cGAS protein;

(ii) logic for design a candidate cGAS inhibitor based on the binding ofthe cGAS protein to the chemical known to bind cGAS protein;

(iii) logic for determining an information regarding a binding of thecGAS protein to the candidate cGAS inhibitor; and

(iv) logic for making a conclusion regarding a cGAS inhibitoryproperties of the candidate cGAS inhibitor based on the determinationstep of step (iii).

In some embodiments, the present invention provides an electronic signalor carrier wave that is propagated over the internet between computerscomprising a set of information for a general purpose computer having auser interface comprising a display unit, the set of informationcomprising a computer-readable storage medium containing a set ofinformation for a general purpose computer having a user interfacecomprising a display unit, the set of information comprising:

(i) logic for inputting an information regarding a binding of a cGASprotein to a chemical known to bind cGAS protein;

(ii) logic for designing a candidate cGAS inhibitor based on the bindingof the cGAS protein to the chemical known to bind cGAS protein;

(iii) logic for determining an information regarding a binding of thecGAS protein to the candidate cGAS inhibitor; and

(iv) logic for making a conclusion regarding a cGAS inhibitoryproperties of the candidate cGAS inhibitor based on the determination ofstep (iii).

EXAMPLES

The following coordinates have been deposited in the RCSB Protein DataBank, with which the skilled artisan will be familiar, and correspond toTables 1-7 referenced herein. See also Gao et al. Cell 153, 1094-1107(2013), including supplementary materials, the entire contents of whichare hereby incorporated by reference herein. Furthermore, in the contextof the ensuing FIGS. 1-4, 7, and 8-11, the data presented in Tables 1-7of U.S. provisional patent application No. 61/819,369, filed May 3,2013, is hereby incorporated by reference.

TABLE E1 Sample PDB code rcsb code ¹ Table cGAS 4K8V RCSB079037 1 cGAS +DNA 4K96 RCSB079048 2 cGAS + DNA + ATP 4K97 RCSB079049 3 cGAS + DNA +5′-pppG(2′,5′)pG 4K98 RCSB079050 4 cGAS + DNA + 5′-pppdG(2′,5′)pdG 4K99RCSB079051 5 cGAS + DNA + 5′-pG(2′,5′)pA 4K9A RCSB079052 6 cGAS + DNA +c[G(2′,5′)pA(3′,5′)p] 4K9B RCSB079053 7 ¹ One method of accessing theRCSB Protein Data Bank is online at www.rcsb.org.

Example 1 Crystal Structures

Protein Expression and Purification

The gene encoding mouse cGAS was purchased from Open Biosystems Inc. Thesequences corresponding to full-length and residues 147-507 of cGAS wereinserted into a modified pRSFDuet-1 vector (Novagen), in which cGAS wasseparated from the preceding His₆-SUMO tag by an ubiquitin-like protease(ULP1) cleavage site. The gene sequences were subsequently confirmed bysequencing. The fusion proteins were expressed in BL21 (DE3) RIL cellstrain. The cells were grown at 37° C. until OD600 reached approx. 0.6.The temperature was then shifted to 18° C. and the cells were induced byaddition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culturemedium at a final concentration of 0.3 mM. After induction, the cellswere grown overnight. The fusion protein was purified over a Ni-NTAaffinity column. The His₆-SUMO tag was removed by ULP1 cleavage duringdialysis against buffer containing 40 mM Tris-HCl, 0.3 M NaCl, 1 mM DTT,pH 7.5. After dialysis, the protein sample was further fractionated overa Heparin column, followed by gel filtration on a 16/60 G200 Superdexcolumn. The final sample of cGAS (full-length) and cGAS (147-507)contain about 30 mg/ml protein, 20 mM Tris, 300 mM NaCl, 1 mM DTT, pH7.5. The Se-methionine substituted protein was expressed inSe-methionine (Sigma) containing M9 medium and purified using the sameprotocol used for the wild-type protein. All the mutants were cloned andpurified using the same protocol as used for preparation of thewild-type protein.

Crystallization

For crystallization of cGAS (147-507) in the free state, the protein wasfirst diluted into about 15 mg/ml and then mixed with equal volumereservoir solution (0.1 M HEPES, 0.1 M MgAc₂, 20% PEG3350, pH 7.6) at 4°C. by using hanging drop vapor diffusion method.

For cGAS (147-507)-dsDNA binary complex, the sample was prepared bydirect mixing protein with a 16-bp DNA (1-nt 5′-overhang at either end:upper strand 5′-AAATTGCCGAAGACGAA-3′; lower strand5′-TTTCGTCTTCGGCAATT-3′) in a 1:1.2 molar ratio. The crystals weregenerated by hanging drop vapor diffusion method at 20° C., from dropsmixed from 1 μl of cGAS-dsDNA solution and 1 μl of reservoir solution(0.1 M MES, 8% MPD, pH 6.6). The crystals of Se-methionine substitutedcGAS (147-507) in complex with dsDNA were grown under the samecondition.

The cGAS (147-507)-dsDNA-ATP, cGAS (147-507)-dsDNA-GTP, and cGAS(147-507)-dsDNA-3′-dGTP ternary complexes were prepared by mixingprotein with dsDNA in a 1:1.2 molar ratio, and then incubated in thepresence of ATP/GTP/3′-dGTP (5 mM) and MgCl₂ (10 mM) for 0.5 h at roomtemperature. The crystals for cGAS (147-507)-dsDNA-ATP complex weregenerated by hanging drop vapor diffusion method at 20° C., from dropsmixed from 1 μl of cGAS-dsDNA-ATP solution and 1 μl of reservoirsolution (0.1 M HEPES, 0.2 M CaAc₂, 20% PEG300, pH 7.7). For cGAS(147-507)-dsDNA-GTP and cGAS (147-507)-dsDNA-3′-dGTP complexes, thecrystals were generated by sitting drop vapor diffusion method at 20°C., by mixing equal volume reservoir solution (for GTP: 0.1 M NaAc, 10%MPD, pH 5.0; for 3′-dGTP: 0.1 M NaAc, 12% MPD, pH 5.2) with the samples.

The cGAS (147-507)-dsDNA-GMP+ATP and cGAS (147-507)-dsDNA-GTP+ATPternary complexes were prepared by mixing protein with dsDNA in a 1:1.2molar ratio, and then incubated with GMP/GTP (5 mM), ATP (5 mM) andMgCl₂ (10 mM) for 0.5 h at room temperature. The crystals for cGAS(147-507)-dsDNA-GMP+ATP complex were generated by sitting drop vapordiffusion method at 20° C., from drops mixed cGAS-dsDNA-GMP+ATP solutionwith equal volume reservoir solution (0.1 M MES, 40% MPD, pH 6.0). Thecrystals for cGAS (147-507)-DNA-GTP+ATP complex were generated over twoweeks by sitting drop vapor diffusion method at 20° C., by mixing equalvolume reservoir solution (0.1 M HEPES, 0.2 M MgCl₂, 30% PEG300, pH 7.5)with the sample.

Structure Determination

The heavy atom derivative crystal of the free state was generated bysoaking in a reservoir solution with 5 mM thimerosal for 24 h. Thediffraction data sets for cGAS (147-507) in free state (both native andHg-derivative) and DNA-bound state (both native and Se-derivative) werecollected at the Brookhaven National Laboratory. The data sets for allthe ternary complexes were collected at the Advanced Photo Source (APS)at the Argonne National Laboratory. The diffraction data were indexed,integrated and scaled using the HKL2000 program (Otwinowski and Minor,1997). The structure of Hg-substituted cGAS (147-507) in free state andSe-substituted cGAS (147-507) in DNA bound state were both solved usingsingle-wavelength anomalous dispersion method as implemented in theprogram PHENIX (Adams et al., 2010). The model building was carried outusing the program COOT (Emsley et al., 2010) and structural refinementwas carried out using the program PHENIX (Adams et al., 2010). Thestatistics of the data collection and refinement for free and binarystructures are shown in Table S1. The structures of all the ternarycomplexes were solved using molecular replacement method in PHASER(McCoy et al., 2007) using the binary structure as the search model. Themodel building was conducted using the program COOT (Emsley et al.,2010) and structural refinement was conducted using the program PHENIX(Adams et al., 2010). The statistics of the data collection andrefinement are shown in Table S2 and S3.

TABLE S1 Data collection and refinement statistics for structures ofcGAS in free and DNA bound state Crystal cGAS cGAS + DNA Beam lineNSLS-29X NSLS-29X Wavelength 0.9790 0.9790 Space group P2₁ C2 Unit cella, b, c (Å) 86.6, 84.1, 124.7 181.9, 93.8, 75.5 α, β, γ (°) 90.0, 92.7,90.0 90.0, 97.7, 90.0 Resolution (Å)    50-2.0 (2.07-2.00)^(a)    50-2.1(2.18-2.10)^(a) R_(merge) 0.179 (0.493) 0.089 (0.511) I/σ (I) 15.7(3.6)  15.8 (3.1)  Completeness (%) 99.3 (98.6) 99.8 (100)  Redundancy7.6 (7.7) 5.5 (5.3) Number of unique 118611 74352 reflectionsR_(work)/R_(free) (%) 17.5/20.8 20.2/22.6 Number of non-H atomsProtein/DNA 11957 7257 Water 1357 722 Ion 4 2 Average B factors (Å²)Protein 34.01 40.90 DNA 68.30 Water 35.56 43.80 Ion 13.80 56.96 R.m.s.deviations Bond lengths (Å) 0.010 0.003 Bond angles (°) 1.207 0.947^(a)Highest resolution shell (in Å) shown in parentheses.

TABLE S2 Data collection and refinement statistics of ternary complexesof cGAS and dsDNA with ATP, GTP, and 3′-dGTP cGAS + DNA + cGAS + DNA +cGAS + DNA + GTP 3′-dGTP Crystal ATP [5′-pppG(2′,5′)pG][5′-pppdG(2′,5′)pdG] Beam line APS-24ID-C APS-24ID-E APS-24ID-EWavelength 0.9823 0.9792 0.9792 Space group /222 /222 /222 Unit cell a,b, c (Å) 86.2, 99.4, 131.5 85.4, 97.9, 133.5 85.1, 97.6, 131.4 α, β, γ(°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å)   50-2.4 (2.54-2.41)^(a)    50-1.9 (2.04-1.94)^(a)    50-2.0(2.05-1.95)^(a) R_(merge) 0.079 (0.577) 0.067 (0.643) 0.079 (0.700) I/σ(I) 14.2 (2.4)  20.5 (2.2)  17.5 (2.3)  Completeness (%) 99.6 (97.8)98.9 (93.5) 100 (100) Redundancy 6.3 (6.3) 9.1 (5.6) 10.7 (10.7) Numberof unique reflections 22099 41225 40199 R_(work)/R_(free) (%) 19.3/23.615.7/19.9 17.6/21.2 Number of non-H atoms Protein/DNA 3492 3521 3521Water 147 363 318 Ion 3 3 3 Other ligands 31 55 53 Average B factors(Å²) Protein 51.83 35.77 38.17 DNA 83.79 64.04 64.83 Water 44.70 40.6840.78 Ion 35.56 21.95 35.32 Other ligands 58.44 32.66 43.53 R.m.s.deviations Bond lengths (Å) 0.007 0.018 0.008 Bond angles (°) 1.1902.183 1.718 ^(a)Highest resolution shell (in Å) shown in parentheses.

TABLE S3 Data collection and refinement statistics of ternary complexesof cGAS and dsDNA with GMP + ATP and GTP + ATP cGAS + DNA + cGAS + DNA +GMP + ATP GTP + ATP Crystal [5′-pG(2′,5′)pA] c[G(2′,5′)pA(3′,5′)p] Beamline APS-24ID-C APS-24ID-E Wavelength 0.9795 0.9792 Space group /222/222 Unit cell a, b, c (Å) 85.4, 98.0, 131.3 85.3, 98.3, 130.0 α, β, γ(°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å)    50-2.3(2.39-2.26)^(a)    50-2.3 (2.38-2.26)^(a) R_(merge) 0.059 (0.303) 0.084(0.904) I/σ (I) 31.5 (4.8)  17.6 (2.4)  Completeness (%) 98.3 (89.2) 100(100) Redundancy 12.1 (5.5)  9.7 (9.9) Number of unique 25590 25950reflections R_(work)/R_(free) (%) 16.8/21.0 17.6/21.8 Number of non-Hatoms Protein/DNA 3521 3518 Water 209 189 Ion 1 1 Other ligands 46 45Average B factors (Å²) Protein 43.30 48.35 DNA 74.18 71.50 Water 43.8643.94 Ion 30.20 25.89 Other ligands 54.77 89.75 R.m.s. deviations Bondlengths (Å) 0.005 0.010 Bond angles (°) 1.433 1.933 ^(a)Highestresolution shell (in Å) shown in parentheses.Structure of Cyclic GMP-AMP Synthase (cGAS)

We have solved the 2.0 Å crystal structure of cGAS (construct 147-507)(FIG. 8A) in the free state (FIGS. 1A and 8B). The protein adopts abilobal scaffold with mixed α/β topology (FIG. 1A; x-ray statistics inTable S1) characteristic of members of the nucleotidyltransferasesuperfamily. A DALI search identified ILF2/NF45, which contains anucleotidyltransferase fold (PDB: 4AT8) (Wolkowicz and Cook, 2012), asmost closely resembling the fold of cGAS, with a Z score of 15.1 andr.m.s.d of 3.8 Å. In addition, the free state of human oligoadenylatesynthetase 1 (OAS1) (PDB: 1PX5) (Hartmann et al. 2003) exhibited a Zscore of 13.3 and a r.m.s.d. of 4.1 Å (comparison of cGAS and OAS1 inthe free state in stereo in FIG. 8C).

Structure of Binary Complex of cGAS with Bound dsDNA

We have cocrystallized cGAS bound to a 16-bp complementary dsDNA (plus1-nt 5′-overhang at either end) and solved the structure of the binarycomplex at 2.1 Å resolution (x-ray statistics in Table S1). Thestructure of the binary complex is shown in FIG. 1B. The majority of theintermolecular contacts in the binary cGAS-dsDNA complex (summarized inFIG. 1C) are between cGAS and the sugar-phosphate backbone of the DNA(FIGS. 9A, B), with only one base-specific contact (FIG. 9B). Thesuperposed structures of cGAS in the free (light gray) and dsDNA-bound(dark gray) states are shown in FIG. 1D. There are large conformationalchanges on formation of the binary dsDNA complex as can be seen within aβ-sheet segment containing catalytic Glu211, Asp213 and Asp307 residues(FIG. 1E), as well for loop and helical segments within the catalyticpocket containing Ser199 (FIG. 1F). Thus, a β-sheet segment shifts by5.1 Å on complex formation (FIG. 9C), as does Arg161 involved inbase-specific recognition by 9.2 Å (FIG. 9D), as do Tyr and Lys residueswithin loop segments by up to 17.6 Å (FIG. 9E). Equally important, avery narrow entrance leads to the catalytic pocket for cGAS in the freestate (FIG. 1G), while this entrance widened significantly in the binarycomplex with DNA (FIG. 1H). The cGAS fold in the dsDNA bound state issimilar to that reported recently for the OAS1 in the dsRNA bound stateplus 2′-dATP (PDB: 4IG8) (Donovan et al. 2013) (comparison of proteinsin complexes in stereo in FIG. 9D; Z score of 18.2 and r.m.s.d. of 3.2 Åbetween the two protein folds).

Structure of Ternary Complex of cGAS with dsDNA and Bound ATP

We cocrystallized cGAS bound to dsDNA and ATP and solved the structureof the ternary complex at 2.4 Å resolution. The ATP is bound in thecatalytic pocket positioned within the interior of the cGAS in theternary complex (FIG. 2A). There is close superposition of the binarycomplex of cGAS and dsDNA with the ternary complex containing bound ATPas shown in FIG. 2B, with essentially minimal conformational changes ineither the β-sheet segment carrying the catalytic acidic residues (FIG.2C) or the loop and helix segments forming the catalytic pocket (FIG.2D) on ternary complex formation. The only notable change is themovement of the side chain of Glu211 towards the other two acidicresidues in the ternary complex (FIG. 2C). The triphosphate group of ATPis hydrogen bonded to polar side chains (Ser199, Ser420 and Lys402),while two bound cations (tentatively assigned to Mg²⁺) serve a bridgingrole for interactions between the triphosphate and the side chains ofcatalytic acidic residues (Glu211, Asp213 and Asp307) (FIG. 2E). Inaddition, the adenine ring of bound ATP stacks over Tyr421 in onedirection and partially over the guanidinium group of Arg364 in theother direction (FIG. 2F).

It should be noted that we observe additional weak electron density(dark gray contours in FIG. 2G) that is unaccounted for at this time inthe 2.4 Å structure of the ternary complex. The additional density couldbe either water molecules or an AMP molecule with modest (30%)occupancy. A view of the bound ATP looking into the catalytic pocket ofthe ternary complex is shown in FIG. 2H.

Structure of Ternary Complex of cGAS with dsDNA and Bound5′-pppG(2′,5′)pG

We cocrystallized cGAS bound to dsDNA and GTP and solved the structureof the ternary complex at 1.9 Å resolution. The structure of the ternarycomplex is shown in FIG. 3A (x-ray statistics in Table S2). Notably,phosphodiester bond formation has occurred in the catalytic pocketyielding the bound ligand 5′-pppGpG (shown positioned in the catalyticpocket in space-filling representation in FIG. 3A). Importantly, minimalconformational changes occurred on proceeding from the binary complex tothe ternary complex with bound 5′-pppGpG (FIGS. 10A-C).

Strikingly, the GpG linkage of 5′-pppGpG is 2′,5′ rather than theanticipated 3′,5′, with the first and second G residues in additionadopting syn and anti glycosidic torsion orientations, respectively(FIG. 10D). The triphosphate group of 5′-pppG(2′,5′)pG is coordinated totwo cations (FIG. 3B), with the first G stacked on Tyr421, while thesecond G uses its Watson-Crick edge to hydrogen bond with polar sidechains (Thr197, Ser366 and Ser368), and its Hoogsteen edge to hydrogenbond with Arg364 (FIG. 3C). The observed 5′-pppG(syn)(2′,5′)pG(anti)topology can be traced with a high degree of confidence because of theclear density observed for this intermediate of the reaction in the 1.9Å structure of the ternary complex (2Fo-Fc map in FIG. 3D, with twoviews of the Fo-Fc omit map shown in FIG. 10E). A view of the bound5′-pppG(2′,5′)pG looking into the catalytic pocket of the ternarycomplex shown is in FIG. 3E. We have superposed the structures of bound5′-pppG(2′,5′)pG (gray) and ATP (dark gray) in their respective ternarycomplexes with cGAS and dsDNA, and observe that the first G of the bound5′-pppG(2′,5′)pG is positioned in the plane of the bound ATP (FIG. 10F).The two bound cations have been tentatively assigned to Mg²⁺ based onomit maps (FIG. 10G) and the octahedral coordination around each cation(FIGS. 10H, I).

We also grew crystals of the ternary complex with 3′-dGTP, and observedformation of the related 5′-pppdG(2′,5′)pdG intermediate (cannot form a3′,5′ linkage) in the 2.1 Å structure of this complex (x-ray statisticsin Table S2).

Structure of Ternary Complex of cGAS with dsDNA and Bound 5′-pG(2′,5′)pA

We have also cocrystallized cGAS in the presence of dsDNA, GMP, and ATPand solved the structure of a complex at 2.3 Å resolution (structuralstatistics in Table S3). By using GMP rather than GTP, we hoped to trapthe intermediate following formation of the first phosphodiester bond,and observed indeed the bound linear product of5′-pG(syn)(2′,5′)pA(anti) (FIGS. 3F, G). No Mg²⁺ cations were observedin the absence of a triphosphate moiety in the product. Notably,attempts at cocrystallization of cGAS with dsDNA, GTP, and AMP onlyyielded crystals that diffracted very poorly (12 Å resolution). Weobserved good superposition of the intermediates5′-pppG(syn)(2′,5′)pG(anti) (in dark gray) and 5′-pG(syn)(2′,5′)pA(anti)(in light gray) as shown in FIG. 3H.

Structure of Ternary Complex of cGAS with dsDNA and Boundc[G(2′,5′)pA(3′,5′)p]

We cocrystallized cGAS with dsDNA, GTP, and ATP and solved the structureof the complex at 2.3 Å resolution. These crystals took two weeks togrow, unlike other crystals mentioned above, that grew within a fewdays. The structure of the ternary complex is shown in FIG. 4A (x-raystatistics in Table S3). Most unexpectedly, the bound small ligand shownin a space-filling representation in FIG. 4A, is a cyclic dinucleotide.Notably, no conformational changes occurred on proceeding from thebinary complex to the ternary complex with bound cyclic dinucleotide,with even the side chain of Glu211 adopting identical orientations(FIGS. 11A-C).

Importantly, we can trace the GpA step in the bound cyclic dinucleotidewithout ambiguity (the 3′-OH of G can be traced) and establish that thislinkage is 2′,5′ (FIG. 11D). On the other hand, the linkage at the ApGstep in the bound cyclic dinucleotide could be either 2′,5′ or 3′,5′based on the observed density, and cannot be assigned with certaintysolely based on structure. We have undertaken the refinement with a3′,5′ linkage at the ApG step based on evidence outlined later andprepared the drawings in FIGS. 4 and 11 with 2′,5′ linkage at the GpAstep and 3′,5′ linkage at the ApG step. We can distinguish G from Abased on the observed density for the 2-amino group of G, and note thatboth adopt anti alignments in the bound cyclic dinucleotide{c[G(2′,5′)pA(3′,5′)p]} (FIG. 11D). The A residue of the boundc[G(2′,5′)pA(3′,5′)p] is stacked on Tyr421 (FIGS. 4B, C), while the Gresidue of the bound c[G(2′,5′)pA(3′,5′)p] is anchored in place throughhydrogen bonding to the side chains of Asp213, Asp307 and Arg364 (FIGS.4B,C). Further, the A and G residues partially stack on each other. The2Fo-Fc electron density for the bound c[G(2′,5′)pA(3′,5′)p] is shown inFIG. 4D, with omit maps shown in FIG. 11E. A view of the boundc[G(2′,5′)pA(3′,5′)p] looking into the catalytic pocket of the ternarycomplex is shown in FIG. 4E, with the c[G(2′,5′)pA(3′,5′)p] boundtowards one end of the opening. We also do not observe bound cations,given that c[G(2′,5′)pA(3′,5′)p] does not contain triphosphates, and theG base directly coordinates with Asp213 and Asp307 (FIG. 4C). We havesuperposed the structures of bound c[G(2′,5′)pA(3′,5′)p] and ATP intheir respective ternary complexes with cGAS and dsDNA, and observe thatthe A of the bound c[G(2′,5′)pA(3′,5′)p] is positioned in the plane ofthe bound ATP (FIG. 11F).

A view of c[G(2′,5′)pA(3′,5′)p] highlighting the 2′,5′ linkage at theGpA step and the 3′,5′ linkage at the ApG step is shown in FIG. 4F. Wenote that in the ternary complex with 5′-pG(2′,5′)pA linear product, itis the G base that stacks on Tyr421 (FIGS. 3G and 4G), while in theternary complex with c[G(2′,5′)pA(3′,5′)p] product, it is the A basethat stacks on Tyr421 (FIGS. 4C, H). Thus, the linear product and cyclicfinal product adopt different alignments within the catalytic pocket.

Example 2 Biochemical Characterization of cGAS Activity

To validate the structural results, we established an activity assayusing thin-layer chromatography (FIG. 11) and monitored cyclicdinucleotide c[G(2′,5′)pA(3′,5′)p] formation from ATP and GTP usingpurified recombinant full-length and truncated cGAS proteins. cGASrequired the presence of dsDNA and Mg²⁺ or Mn²⁺ for activity (FIGS.5A-B). We tested c[G(2′,5′)pA(3′,5′)p] formation as a function of dsDNAlength and found that dsDNA of 36 bp or longer were optimal, yet the 16bp dsDNA used for crystallography elicited some activity (FIG. 13A).Double-stranded RNA, a DNA/RNA duplex, or single-stranded DNA or RNA didnot stimulate cyclic dinucleotide formation (FIG. 5B). The trace amountof c[G(2′,5′)pA(3′,5′)p] detected for the specific ssDNA used in thisexperiment was attributable to a stretch of sequence complementarity,and the substitution of G by 8-oxoguanine (8-oxoG) was sufficient todestabilize its predicted interaction and eliminate the residual cGASactivity. Replacement of guanine by 8-oxoG within the dsDNA did notalter cGAS activity.

We quantified the activity of cGAS to yield c[G(2′,5′)pA(3′,5′)p] undermultiple turnover conditions. Over 78% (s.d.+/−2.6%, n=5) of theoriginal ATP and GTP provided was converted to c[G(2′,5′)pA(3′,5′)p]within 40 min leading to an estimated observed rate constant of 0.19min⁻¹ and involving over 750 turnovers per enzyme molecule.

To determine the order of intermediate formation, we first substitutedGTP by GMP or GDP (FIG. 5C). Both compounds led to the formation of therespective 5′-pG(2′,5′)pA product and 5′-ppG(2′,5′)pA intermediates, andonly 5′-ppG(2′,5′)pA could react further to yield a reduced amount ofthe cylic-dinucleotide. Substitution of ATP by ADP or AMP resulted in noproduct or intermediate formation, with only ADP (with GTP) leading tothe generation of reduced levels of c[G(2′,5′)pA(3′,5′)p].

In order to determine the involvement of 2′ or 3′-hydroxyl (OH) groupsof GTP and ATP for formation of c[G(2′,5′)pA(3′,5′)p], we tested 2′- and3′-deoxyguanosine triphosphate and 2′ and 3′ and deoxyadenosinetriphosphate as substrates for cGAS (FIGS. 5D and 13B). 2′-dGTP wasunable to form a cyclic dinucleotide, unlike 3′-dGTP, indicating thatthe 2′-OH of guanosine was required for the formation of the linkagewith the a-phosphate at the 5′ position of adenosine. In contrast, both2′- and 3′-dATP led to markedly reduced formation of cyclicGA-dinucleotides, although 2′-dATP+GTP yielded noticeably more product.In both cases, we observed accumulation of 5′-pppG(2′,5′)pdA reactionintermediates (FIG. 5D, lanes 2 and 3), which migrated slightly fasterthan the all ribose intermediate (5′-pppG(2′,5′)pA, lane 1).

Example 3 Identification of c[G(2′,5′)pA(3′,5′)p] as the Product Formedby dsDNA-Dependent cGAS Activity

The syntheses and purification of the three isomeric cGAMP molecules,c[G(2′,5′)pA(2′,5′)p] (2′,5′ linkages at GpA and ApG steps) 6,c[G(2′,5′)pA(3′,5′)p] (2′,5′ at GpA step and 3′,5′ at ApG step) 11, andc[G(3′,5′)pA(3′,5′)p] (3′,5′ at GpA and ApG steps) 15 shown in FIG. 14were carried out using procedures previously reported by the Joneslaboratory (Gaffney et al. 2010; Gaffney and Jones, 2012). The identityof the three isomeric cGAMP molecules 6, 11 and 15 (FIG. 14) wasvalidated from heteronuclear NMR analysis using through-bondconnectivities. The experimental NMR data for c[G(2′,5′)pA(3′,5′)p] isoutlined in FIG. 15, with the proton and carbon chemical shifts for allthree isomeric GMP molecules listed in Table S4.

We analyzed the product generated by dsDNA-dependent cGAS activity usingreverse-phase high-performance liquid chromatography (HPLC) and comparedits elution profile to chemically synthesized c[G(3′,5′)pA(3′,5′)p],c[G(2′,5′)pA(2′,5′)p], and c[G(2′,5′)pA(3′,5′)p] compounds (FIGS. 6A and13D). A prominent peak consistently eluted from the HPLC system atprecisely 23.5 min, which corresponded to the elution profile ofc[G(2′,5′)pA(3′,5′)p]. Co-injection with c[G(3′,5′)pA(3′,5′)p] orc[G(2′,5′)pA(2′,5′)p] demonstrated that the cGAS reaction product doesnot co-elute, unlike co-injection with chemically synthesizedc[G(2′,5′)pA(3′,5′)p].

To demonstrate that the in vitro produced cyclic dinucleotide matchedthe molecule determined crystallographically, we analyzed by HPLC thedissolved cGAS crystals that had been co-incubated with DNA, ATP, andGTP (FIG. 6B). A peak corresponding to c[G(2′,5′)pA(3′,5′)p] wasobserved, distinct from the c[G(2′,5′)pA(2′,5′)p] co-injected referencemolecule as before. Additional unidentified peaks of longer retentiontimes were also seen. Presumably, these unidentified compoundsoriginating from the crystallization buffer and/or additives were notcompletely removed despite washing the crystals prior to HPLC analysis.

In addition, cGAS-generated c[G(2′,5′)pA(3′,5′)p] was purified by HPLCand subjected to one-dimensional NMR analysis. Its NMR spectrum in thesugar H1′ region is identical to that of chemically synthesized standardc[G(2′,5′)pA(3′,5′)p] and distinct from chemically synthesizedc[G(2′,5′)pA(2′,5′)p] and c[G(3′,5′)pA(3′,5′)p] (FIG. 6C). Thus, bothHPLC (FIG. 6A) and NMR (FIG. 6C) independently validate that the productgenerated by cGAS is c[G(2′,5′)pA(3′,5′)p].

Example 4 Functional Analysis of cGAS Mutant Proteins

We next assayed the biochemical and functional consequences of mutationson cGAS in its capacity to form c[G(2′,5′)pA(3′,5′)p] in vitro and tostimulate the type I interferon pathway in cells. We generatedalanine-substitution mutants corresponding to amino acid residues thatthe co-crystal structures revealed to be involved in dsDNA binding orcGAS activity. For in vitro cGAS activity assays, we generated andpurified six recombinant mutant cGAS forms; four were predicted toeliminate dsDNA-binding and two point mutant proteins were substitutedwith alanine at potentially key catalytic residues. Incubation of DNAwith mutant cGAS proteins led to little or no c[G(2′,5′)pA(3′,5′)p]formation for all but two mutants (R161A, S199A, FIG. 7A).

To assess the impact on cGAS function in cells, we generated additionalalanine mutants of cGAS for expression in mammalian cells. Thefull-length cGAS mutants together with STING and an IFN-β luciferasereporter were transiently expressed in HEK 293 cells. In this assay cGASis engaged by the co-transfected DNA plasmids, and WT cGAS expressionresulted in close to 15-fold enhanced luciferase activity compared to acontrol plasmid (FIG. 7B). Single mutations of DNA binding residues,including Arg161 responsible for the only direct interaction with a DNAbase, were not sufficient to impair cGAS activity. However, ablation ofinteractions with two or three consecutive phosphodiesters in either DNAstrand (FIG. 1C, 9B-C) resulted in diminished, or entirely abrogatedcGAS function (FIG. 7B). At the catalytic site, single mutants Glu211,Asp213 or Asp307 affecting the binding of divalent cations (FIGS. 2E,3B) all resulted in non-functional cGAS (FIG. 7C). Furthermore,abrogation of cGAS activity required mutation of both amino acidresidues involved in (i) the binding of ATP (or GTP) gamma phosphate(Lys402, Ser420; FIG. 2E, 3B), (ii) the binding of ATP adenosine(Glu371, Lys424; FIG. 2F), or (iii) the base stacking of ATP andc[G(2′,5′)pA(3′,5′)p] (Arg364, Tyr 421; FIG. 2F, 4B), while singlemutants of these residues only slightly impaired cGAS function (FIG.7C). Gly198 and Ser199 are highly conserved residues that were found toundergo significant conformational changes upon ligand binding (FIG. 1F,2D). Nevertheless, single mutations G198A and S199A did not severelyimpair cGAS function, but the double mutant of these positions was notfunctional (FIG. 13E). Similarly, conversion of the highly mobile Gly198to sterically restricted proline abrogated cGAS activity (FIG. 13E).

Example 5 Studies on Conformational Transitions, Bond Formation, andIntermediates Conformational Transitions in cGAS on Complex Formation

Our structural studies highlight the fact that cGAS undergoes apronounced conformational change upon binding of dsDNA (FIG. 1D),whereby it repositions catalytic residues Glu211, Asp213, Asp307, aswell as Ser199 (FIG. 1E, F), while at the same time opening access tothe catalytic pocket (FIG. 1G, H). In essence, cGAS adopts acatalytically competent conformation only when engaging dsDNA, therebyaccounting for its role as a cytosolic dsDNA sensor. By contrast, onlyminimal conformational changes that are restricted to the side chain ofGlu211 are observed when proceeding from the binary complex of cGAS anddsDNA to the ternary complex with ATP (FIG. 2B-D) and GTP (where thepocket contains off-pathway intermediate 5′-pppG(2′,5′)G; FIG. 10A-C),with no change observed even for the side chain of Glu211 on formationof the ternary complex with ATP+GTP (where the pocket contains productc[G(2′,5′)pA(3′,5′)p]; FIG. 11A-C).

Phosphodiester Bond Formation in Catalytic Pocket

The structural and functional experiments both establishedphosphodiester bond formation in the catalytic pocket after binding ofdsDNA to cGAS, in the absence of any additional components. Thestructural studies on cGAS in the presence of dsDNA and GTP identifiedaccumulation of linear reaction intermediate 5′-pppG(2′,5′)pG (FIG.3B,C), while in the presence GMP and ATP identified accumulation oflinear reaction product 5′-pG(2′,5′)pA. While not wishing to be bound byany particular theory, it is believed that the former intermediate isoff-pathway and therefore impaired for the formation of the secondphosphodiester bond to form a cyclic product, given that the first G issyn, and the distance is long between the 2′-OH (or 3′-OH) of the secondG and the a-phosphate of the triphosphate moiety. Nevertheless, theseresults suggest that formation of c[G(2′,5′)pA(3′,5′)p] is likely tooccur in a stepwise manner, involving formation of sequentialphosphodiester bonds to yield the cyclic dinucleotide product. Bycontrast, structural studies on cGAS in the presence of dsDNA, GTP, andATP resulted in formation of c[G(2′,5′)pA(3′,5′)p] (FIG. 4B, C), withoutaccumulation of an intermediate and consistent with an on-pathwayreaction involving formation of a pair of sequential phosphodiesterlinkages.

Positioning of G and A Residues of Bound c[G(2′,5′)pA(3′,5′)p]

The G and A residues of c[G(2′,5′)pA(3′,5′)p] adopt distinct positionsin the structure of the ternary complex with cGAS and dsDNA. The Aresidue of the bound c[G(2′,5′)pA(3′,5′)p] is stacked on Tyr421 (FIG.4B) and occupies the position of the adenine ring in the ATP complex(FIG. 2F) and the first base in the 5′-pppG(2′,5′)pG (FIG. 3C) and5′-pG(2′,5′)pA (FIG. 3G) complexes. The A residue of boundc[G(2′,5′)pA(3′,5′)p] is not involved in any intermolecular hydrogenbonds and hence could potentially be replaced by even a pyrimidine (C orU) residue. By contrast, the G residue of bound c[G(2′,5′)pA(3′,5′)p],which is partially stacked over the A residue, forms a network ofintermolecular hydrogen bonds involving its Watson-Crick and Hoogsteenedges (FIG. 4B, C) and cannot be replaced by any of the other threebases (C, A and U). Thus, the cGAS-binding pocket has distinctrecognition elements that distinguish between G and A and hence can bindc[G(2′,5′)pA(3′,5′)p] in a unique orientation.

In agreement with the crystallographic data, the biochemical resultsindicate a strong preference for GTP, consistent with the elaborateamino acid interactions observed in the structure between cGAS and thisbase. Incubation of cGAS with GTP alone, as well as GTP plus either CTPor UTP, can lead to cyclical dinucleotide formation (FIG. 13C). WhileATP alone does not yield any cyclic or intermediate products, incubationwith UTP results in some cyclic product formation, suggesting that UTPcan also substitute for GTP albeit at a much reduced reaction rate.Together, these findings indicate that cGAS has more relaxedrequirements for the second nucleotide compared to the first guanosine.

Structural Comparison of Linear 5′-pG(2′,5′)pA and Cyclicc[G(2′,5′)pA(3′,5′)p] Product

We observe a striking difference in alignment within the catalyticpocket between the off-pathway linear 5′-pG(syn)(2′,5′)pA(anti) product(FIG. 3F, G) and cyclic 5′-pG(anti)(2′,5′)A(anti) product (FIG. 4B, C).In the former case, it is the G base that stacks over Tyr421 (FIG. 3G),while in the latter it is the A base that stacks over Tyr421 (FIG. 4C).The two alignments are compared in stereo where the linear5′-pG(2′,5′)pA is shown in FIG. 4G and the cyclic c[G(2′,5′)pA(3′,5′)p]in FIG. 4H. This implies that the intermediate may have to rearrange itsorientation by a complete flip-over within the catalytic pocket prior tothe cyclization reaction. This may not be too surprising since judgingfrom the three basic (Glu211, Asp213 and Asp307) and one polar (Ser199)amino acid lining the catalytic pocket, there is only a single set ofcatalytic residues and hence following the first phosphodiester bondformation, the intermediate may have to realign so as to facilitate thesecond phosphodiester bond formation to complete cyclization.

Phosphodiester Linkages

A clear assumption in the earlier studies leading to the identificationof cyclic GAMP as a second messenger generated by the cytoplasmic dsDNAsensor cGAS (Sun et al. 2013; Wu et al. 2013) was that bothphosphodiester linkages were of a 3′,5′ nature. Such 3′,5′ linkages havebeen observed previously in structures of bacterial second messengerc-di-GMP bound to both STING (Yin et al 2010; Ouygang et al. 2012; Huanget al. 2012; Shu et al. 2012) and riboswitches (Smith et al. 2009;Kulshina et al. 2009). Nevertheless, the mass spectroscopic approachutilized (Wu et al. 2013) cannot distinguish between 3′,5′ and 2′,5′linkages for one or both phosphodiester bonds of cyclic GAMP.

The first indication of a 2′,5′ linkage emerged from the structure ofcGAS, dsDNA and GTP, where an off-pathway product formed in thecatalytic pocket, exhibited a 5′-pppG(2′,5′)pG linkage (FIG. 3B, C). Inaddition, pG(2′,5′)pA was observed in the structure of cGAS, dsDNA andGMP+ATP (FIG. 3F, G). More importantly, a 2′,5′ linkage was alsoobserved for the GpA step of the bound c[G(2′,5′)pA(3′,5′)p] product inthe catalytic pocket in the structure of cGAS, dsDNA and GTP+ATP (FIG.4B, C).

Our initial biochemical analyses indicated that the 2′,5′ linkagebetween GTP and ATP occurs first, prior to the cyclization of theadenosine back to guanosine. This evidence was further supported by theobservation that incubation of 2′-dATP with 2′-dGTP could not react toform any cyclic reaction products (FIG. 13). In the second step,formation of a cyclical dinucleotide via the 2′ or 3′ OH of adenosinecan proceed even when the other position is blocked through removal ofoxygen, although there was an observable preference for utilization of2′-dATP. Cyclic dinucleotide production in either case was veryinefficient, suggesting that both positions may participate in theformation of a transition state for efficient phosphate hydrolysis andcyclization. This perplexing result, combined with ternary structuraldata concerning the connection from adenosine to guanosine, prompted usto further examine whether cGAS would ultimately have a preference forgenerating a 2′,5′ or 3′,5′ link for cyclization.

We observed a single HPLC peak, distinct from two cyclic-GA dinucleotidereference molecules (either both 2′,5′ or both 3′,5′ linkages) andcoincident with c[G(2′,5′)pA(3′,5′)p], as the product of dsDNA-dependentcGAS activity (FIG. 6A). This conclusion was validated from anindependent NMR study (FIG. 6C). While biological production of cyclicdinucleotides appears evolutionarily conserved from prokaryotes toeukaryotes, their formation based on the chemical linkages is distinct.In the case of cGAS, formation of c[G(2′,5′)pA(3′,5′)p] appears similarto the 2′,5′ oligoadenylates generated by OAS but also to the 3′,5′dinucleotide linkages created by bacterial cyclases (Sadler andWilliams, 2008; Kodym et al. 2009; Donovan et al. 2013).

Potential Benefits of 2′,5′ (GA Step) and 3′,5′ (AG Step) Linkages inc[G(2′,5′)pA(3′,5′)p]

It is not clear why cGAS prefers to generate both a 2′,5′ (GpA step) and3′,5′ (ApG step) cyclic GA-dinucleotide. A 2′,5′ phosphodiester bond isuncommon and few nucleases are known to be able to hydrolyze such alinkage (Kubota et al. 2004). Without wishing to be bound by anyparticular theory, 2′,5′ linkages might promote greater stability incells to enable effective transduction of the second messenger, but the3′,5′ linkage may facilitate its breakdown by numerous conventionalendonucleases to prevent prolonged interferon response. Taken together,our structure and functional studies have identified the chemical natureof metazoan cGAMP, highlighting the role of 2′,5′ linkages in secondmessengers that activate the type I interferon pathway.

Implications of cGAS dsDNA-Binding Mutants

Structural studies have identified intermolecular protein-DNA contactson formation of the cGAS-dsDNA complex (FIG. 1C). Since these areprimarily of an electrostatic nature and involve non-specificrecognition of the DNA phosphodiester backbone, they have beenclassified into three sets of triple mutants, with the in vitro (FIG.7A) and cellular assays (FIG. 7B) establishing complete loss in activityand ability to stimulate interferon production for the S165A, N172A,K372A triple mutant, the N196A, Y200A, K372A triple mutant and theR158A, R161A, K395A triple mutant, and partial loss in activity for theS165A, N172A, Y200A triple mutant. This reinforces the importance ofcomplex formation between cGAS and dsDNA for the catalytic activity ofcGAS.

Implications of cGAS Catalytic Pocket Mutants

Structural studies of ternary complexes of cGAS and dsDNA with boundNTPs have identified Glu211, Asp213, Asp307 as important catalyticresidues for phophodiester bond formation. All three catalytic acidicresidues are functionally dead on replacement by Ala as observed ineither in vitro (FIG. 7A; Glu211A) or cellular (FIG. 7C, all threecatalytic residues) assays, while the S199A mutation retainedsubstantial activity. Tyr421 is involved in stacking interactions withA, while Arg364 is hydrogen bonded with G in the cGAMP ternary complex(FIG. 4B, C). Dual mutation of Y421A, R364A result in loss in themajority of activity in cellular assays (FIG. 7C).

Role of Divalent Cations

Structural studies of ternary complexes of dsDNA-bound cGAS with ligandshave shown that the triphosphate moieties of ATP (FIG. 2E, F) and5′-pppG(2′,5′)pG (FIG. 3B, C) are coordinated to a pair of cations(tentatively assigned to Mg²⁺). Indeed, functional studies havehighlighted the importance of divalent cations to phosphodiester bondformation. Omission of divalent cations or use of EDTA preventedc[G(2′,5′)pA(3′,5′)p] formation, whereas Mg²⁺ and Mn² promoted cGASactivity (FIG. 5A).

Comparison with Cytoplasmic dsRNA Sensor OAS1

In a parallel study to our contribution, structural studies andbiochemical assays have been recently reported on the characterizationof the dsRNA sensor human oligoadenylate synthetase 1 (OAS1) whichpolymerizes ATP into linear 2′,5′-linked oligoadenylate (Donovan et al.2013). The crystallographic studies unequivocally demonstratedconformational transitions in OAS1 on proceeding from the free state(Hartmann et al. 2003) to the ternary complex with bound dsRNA and2′-dATP (Donovan et al. 2013), which follow a similar pattern to thoseobserved by us in this study for complex formation of cGAS with dsDNAand bound ligands. Thus, three catalytic Glu residues of OAS1 arebrought into close proximity on formation of the ternary complex withdsRNA and 2′-dATP, thereby creating the coordination geometry forbinding of two Mg²⁺ ions and 2′-dATP (Donovan et al. 2013), similar towhat we observe for the cGAS system. Given that the only availablestructures were for free OAS1 (Hartmann et al. 2003) and its ternarycomplex with dsRNA and bound ligand (Donovan et al. 2013), these authorswere not in a position to determine how the conformational transitionwas partitioned between steps reflecting conversion from free OAS1 tothe binary complex with dsRNA and conversion from the binary complex tothe ternary complex in the presence of 2′-dATP. Our results on thecytosolic dsDNA sensor cGAS suggest that the major conformationaltransition will most likely be restricted for the step involvingconversion of OAS1 from the free state to the dsRNA-binding complex,with minimal changes on addition of 2′-dATP to form the ternary complex.

In addition to similarities mentioned above, there are also differencesin protein-nucleic acid recognition principles between the cGAS dsDNAsensor (our study) and the OAS1 dsRNA sensor (Donovan et al. 2013), inthat cGAS targets the sugar-phosphate backbone of dsDNA within a centralsegment of the dsDNA duplex (FIG. 1C), while OAS1 targets thesugar-phosphate backbone of dsRNA by contacting two minor groovesegments separated by 30 Å (Donovan et al. 2013). The helical parametersof dsRNA and dsDNA are very distinct, and different recognitionprinciples are used in protein-dsRNA (reviewed in Lunde et al. 2007) andprotein-dsDNA (reviewed in Huffman and Brennan, 2002) complexes.Nevertheless, common principles are utilized to generate the criticalcatalytic site architecture, which in turn couples nucleic acidrecognition (dsRNA or dsDNA in the cytoplasm) with the cascade ofdownstream events leading to an antiviral state including type Iinterferon response (cGAS) and RNase L activation (OAS1).

Further, the formation of linear 2′,5′-linked iso-RNA mediated by OAS1parallels the formation of c[G(2′,5′)pA(3′,5′)p] containing 2′,5′linkage at the GpA step by cGAS. Thus, unlike earlier sole emphasis on3′,5′ linkages as observed previously for bacterial second messengerc-di-GMP (reviewed in Romling et al. 2013), we highlight that themetazoan second messenger c[G(2′,5′)pA(3′,5′)p] utilizes mixed linkagesinvolving 2′,5′ at the GpA step and 3′,5′ at the ApG step.

cGAS Contains a Single Active Site for Step-Wise Phosphodiester BondFormation

Previous studies have established that diguanylate cyclase PleD forms ahead-to-tail homodimer to form a reaction center at its interface, sothat the intermediate does not have to change its orientation on thepathway to form c-di-GMP (Chan et al. 2004). By contrast, in our currentstudies of ternary complexes of cGAS, dsDNA and bound ligands, weobserve no evidence for dimer or higher order oligomer formation in thecrystal. Further, the ligand-binding pocket in our structures is buriedwithin the cGAS topology and is not located on the surface, as it is inPleD (Chan et al. 2004).

Indeed, cGAS contains a single active site for the sequentialphosphodiester bond formation steps, a feature quite remarkable giventhat the ligands are GTP and ATP and that the GpA linkage which formsfirst is 2′-5′ and the ApG linkage which forms second is 3′,5′,resulting in generation of c[G(2′,5′)pA(3′,5′)p]. Without wishing to bebound by any particular theory, we outline a model for formation ofc[G(2′,5′)pA(3′,5′)p] from GTP and ATP within the single catalyticpocket of dsDNA-bound cGAS in FIG. 7D. In this model, the first stepinvolves formation of a 5′-pppGpA intermediate followed in the secondstep by formation of c[G(2′,5′)pA(3′,5′)p]. Note, also that the boundligand is believed to undergo two flip-overs on the pathway toc[G(2′,5′)pA(3′,5′)p] formation.

Implications for the Di-Nucleotide Cyclase DncV

In an earlier study, the bacterial dinucleotide cyclase DncV was shownto generate cyclic GMP-AMP (cGAMP) (Davies et al. 2012). This firstreport on formation of cGAMP raises the interesting question as to thenature of the pair of phosphodiester linkages in this bacterial system.

Example 6 NMR Spectral Analysis of Synthesized cGAMP Linkage Isomers

Lyophilized cGAMP linkage isomers were dissolved in 99.9% D₂O in 10 mMK₂HPO₄—KH₂PO₄ (pH 6.6) buffer. All NMR experiments are conducted at 35°C. on a Bruker 900 MHz spectrometer at New York Structural BiologyCenter. Resonance assignments are made based on HMBC (2 s recyclingdelay, 0.8 s ¹H acquisition time, 20 ms ¹³C acquisition time,phase-insensitive ¹³C acquisition, and anti-phase ¹H detection withabsolute value mode processing), double-quantum filtered COSY (2 srecycling delay, 0.8 s direct acquisition time, 12 ms indirectacquisition time), and HSQC experiments (1 s recycling delay, 48 ms ¹Hacquisition time, 20 ms ¹³C acquisition time). The 1D proton spectrawith water presaturation are accumulated over 8 scans for thesynthesized cGAMP linkage isomers standards and 128 scans for thebio-enzymatically produced cGAS reaction.

Example 7 Thin Layer Chromatography (TLC) Analysis

Preparation of Oligonucleotides for TLC Assays

Oligonucleotides used for biochemical assays of cGASnucleotidyltransferase activity are listed in Table S6.Oligodeoxynucleotides were synthesized in-house using a 3400 DNAsynthesizer (Applied Biosystems), oligoribonucleotides were purchased(Dharmacon). Double-stranded DNA, RNA, and DNA/RNA duplexes wereannealed in 70 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 5 mM DTT, at equimolarconcentrations by incubation initiated at 95° C. followed by a 0.1° C.decrease per second to 25° C. in a Peltier thermoycler (MJ Research),and verified for annealing by agarose gel electrophoresis prior to use.

TLC Analysis of c[G(2′,5′)pA(3′,5′)p] Formation

Purified recombinant full length (fl, amino acids 1-507) and truncated(tr, amino acids 147-507) murine cGAS, including truncated mutantversions 1-6, were incubated in 20 μL reactions containing: 1 μM cGAS,3.3 μM dsDNA, 5 mM MgCl₂, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5 at 25° C.,1 mM DTT, 10% glycerol, 1 mM each of nucleotides (typically ATP andGTP), and α³²P or γ³²P radiolabelled NTPs or dNTPs at 37° C. for 40 min.Reactions were stopped by addition of 20 μL of 50 mM EDTA. 2 μL ofreaction solution was spotted onto high-performance TLC plates (HPTLCsilica gel, 60 Å pores, F₂₅₄, 10×10 cm, cat #1.05628.0001, EMDMillipore) and products were separated with Solvent 1(NH₄HCO₃:C₂H₅OH:H₂O [0.2 M:30%:70%], w:v:v) or 2 (NH₄HCO₃:C₂H₅OH:H₂O[0.025 M:30%:70%], w:v:v) at 25° C. for 1 h. Reaction products werevisualized by UV (254 nm) and phosphorimaging (Typhoon FLA 9500, GEHealthcare). Images were processed using Adobe Photoshop and IllustratorCS5. The TLC conditions used were largely based on a protocolestablished to separate 3′,5′ cAMP (Higashida et al, 2012).

Example 8 Quantitation of cGAS Reaction Products

The yield of c[G(2′,5′)pA(3′,5′)p] generated was calculated bydensitometry analysis of TLC experiments, using FIJI (ImageJ 1.47i) orspectrophotemetrically (absorbance at 260 nm, E₂₆₀=25.4×10³) afterpurification from HPLC. For densitometry analyses, the fraction ofα³²P-labelled c[G(2′,5′)pA(3′,5′)p] over total radioactivity per lane(c[G(2′,5′)pA(3′,5′)p] plus remaining α³²P-labelled ATP or GTP) wascalculated.

Example 9 Preparation of cGAS Reaction Products for High-PerformanceLiquid Chromatography Analysis

In vitro generated c[G(2′,5′)pA(3′,5′)p] reaction products, or washedand dissolved cGAS crystals, were treated with 25 units of Benzonase(Novagen, cat. #70746, Purity>90%) for 30 min at 37° C., heatinactivated for 10 min at 95° C., then centrifuged at 21,000 g for 15min (Sorvall Legend Micro 21R, Thermo Scientific); the supernatant wasused for HPLC analysis. Reaction products, with or without 3-8 nmoles ofchemically synthesized all 3′,5′ cGAMP, all 2′,5′ cGAMP, orc[G(2′,5′)pA(3′,5′)p] were subjected to reverse-phase HPLC analysis(AKTA Purifier, GE Healthcare) using a C18 column (25 cm×4.5 mm, 5 μMpore, Supelco Analytical). Analytes were monitored by UV 260 and 280 nm.A 0-10% solvent B (2 column volume), 10-50% solvent B (2 column volume)two-step linear gradient was used; solvent A (triethylammoniumacetate:acetonitrile:H₂O [0.1 M:3%:97%], w:v:v) and solvent B(methanol:acetonitrile:H₂O [45%:45%:10:], v:v:v).

Preparation of cGAS Reaction Product for 1D NMR Analysis

100 μl of in vitro generated c[G(2′,5′)pA(3′,5′)p] reaction product wasbenzonase and heat treated as before, prior to fractionation by HPLC.Three serial HPLC runs were performed (two 40 μl and one 20 μl reactioninjection), and the peak corresponding to c[G(2′,5′)pA(3′,5′)p] wascollected into a 15 ml falcon tube (approx. 4.5 ml total). Solventremoval was accomplished by vacuum centrifuge (Vacufuge, Eppendorf) for3 days at room temperature until completely dry.

Example 10 Cellular Assays

Generation of cGAS Point Mutants

The murine cGAS CDS was inserted into a modified pMAX-cloning vector(Amaxa, Cologne, Germany). Site-directed mutagenesis was performed usingthe Quikchange method (Agilent, Santa Clara, Calif.) using Pfu Ultra HotStart DNA Polymerase (Agilent) or KOD Hot Start DNA Polymerase (Merck,Darmstadt, Germany). The murine STING CDS and Firefly Luciferase(Promega, Madison, Wis.) were cloned into an EF1-promoter-modifiedpLenti6 (Invitrogen, Carlsbad, Calif.) expression plasmid. pGL3 IFN-betaGluc reporter was obtained from Brian Monks (Institute of InnateImmunity, University of Bonn, Germany). All constructs were verified bysequencing of the CDS.

Luciferase Assay

3×10⁴ HEK293 cells per 96-well were reverse-transfected in triplicateswith a mixture of pGL3-IFNbeta-Gluc (50 ng), pLenti-EF1-Fluc (25 ng),pLenti-EF1-mSTING (25 ng) and cGAS-expression plasmid (25 ng, pMAX-cGASWT or mutants) or Control plasmid pMAX-GFP (Amaxa) using Trans-IT LT1(MirusBio, Madison, Wis.). After 36 h cells were lysed in passive lysisbuffer. Firefly and gaussia Luciferase activities were determined on anEnVision reader (Perkin Elmer, Waltham, Mass.) using the respectivesubstrates D-luciferin and coelenterazine (PJK GmbH, Kleinblittersdorf,Germany). IFNbeta-Gluc values were normalized to constitutive fireflyluciferase values and fold induction was calculated in relation tocontrol-plasmid pMAX-GFP.

Example 11 Synthesis of Cyclic GA-Dinucelotides

Preparation of all 2′,5′-cGAMP (6, FIG. 14), c[G(2′,5′)pA(3′,5′)p] (11,FIG. 14), and all 3′,5′-cGAMP (15, FIG. 14) were carried out using theprocedure previously reported by the Jones laboratory (Gaffney et al.2010; Gaffney and Jones 2012). To adenosine phosphoramidite, 1 or 7,(0.784 g, 0.793 mmol) dissolved in 5 mL of CH₃CN and water (0.028 mL,1.6 mmol, 2 equiv) was added pyridinium trifluoroacetate (0.184 g, 0.95mmol, 1.2 equiv). After 1 min, 6 mL of tert-BuNH₂ was added. Afteranother 10 min, the mixture was concentrated. To the residue dissolvedin 10 mL of CH₂Cl₂ was added H₂O (0.14 mL, 7.9 mmol, 10 equiv), followedby 10 mL of 6% dichloroacetic acid (DCA, 7.5 mmol) in CH₂Cl₂. After 10min, the reaction was quenched by addition of pyridine (1.2 mL, 15 mmol,2 equiv rel to DCA). The mixture was then concentrated, and the residuewas dissolved in 7 mL of CH₃CN and concentrated again.

This process was repeated two more times, the last time leaving the AH-phosphonate, 2 or 8, in 2 mL. To this solution was added a driedsolution of G amidite, 3 or 12 (1.00 g, 1.03 mmol, 1.3 equiv) in 3 mLCH₃CN. After 2 min, anhydrous tert-butyl hydroperoxide 5.5 M in decane(0.43 mL, 2.4 mmol, 3 equiv) was added. After 30 min, 0.20 g of NaHSO₃dissolved in 0.5 mL H₂O was added. The mixture was stirred for 5 min,and then concentrated. The residual oil was dissolved in 14 mL ofCH₂Cl₂, followed by addition of H₂O (0.15 mL, 8.5 mmol, 10 equiv) andthen 14 mL of 6% DCA (9.8 mmol) in CH₂Cl₂. After 10 min, the reactionwas quenched with 9 mL of pyridine. The mixture was concentrated to asmall volume, 25 mL more pyridine was added, and the solution wasconcentrated again, leaving the linear dimer, 4, 9, or 13, in 17 mL. Tothis solution was added5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphinane (DMOCP, 0.54 g of 95%reagent, 2.8 mmol, 3.5 equiv). After 10 min, the reaction was quenchedby addition of H₂O (0.50 mL, 28 mmol, 10 equiv rel to DMOCP), and I₂(0.26 g, 1.0 mmol, 1.3 equiv) was added immediately. After 5 min, themixture was poured into 120 ml, of H₂O containing 0.17 g NaHSO₃. After 5min of stirring, 3.4 g of NaHCO₃ was slowly added. After 5 min more ofstirring, the aqueous solution containing solid was partitioned with 135mL 1:1 EtOAc:Et₂O. The separated aqueous layer was then partitioned withan additional 35 mL of 1:1 EtOAc:Et₂O.

The organic layers containing 5, 10, or 14 were combined andconcentrated to an oil. For 14, the oil was dissolved in 5 mL CH₃CN andthe cyanoethyl group was removed by addition of 5 mL of tert-BuNH₂ for10 min. The residue was purified on a 80 g SiO₂ column, using a gradientof 0 to 25% CH₃OH in CH₂Cl₂ over 50 min. 5 and 10 were directly purifiedon SiO₂ without tert-BuNH₂ treatment. In each case the residue afterpurification was treated with 21 ml, of CH₃NH₂ in anhydrous EtOH (33% byweight, 168 mmol, 212 equiv rel to the amino protecting groups). After 4h at room temperature, the mixture was concentrated to a solid, to which3 mL of pyridine and 1 mL of Et₃N were added. The mixture wasconcentrated to an oil, and this process was repeated two more times toconvert the tert-BuNH₃ ⁺ to the Et₃NH⁺ salt. To the oil was added 1 mLof pyridine, and the flask was placed in an oil bath at 55° C. Et₃N (7.5mL) and Et₃N.3HF (2.6 mL, 48 mmol F⁻, 30 eq rel to each TBS) were addedsimultaneously. The mixture was stirred at 55° C. After 3 h, the flaskwas removed from the oil bath and HPLC grade acetone (70 ml) was slowlyadded to the stirring mixture. After 10 min, the solid was collected byfiltration, washed 5× with 3 mL portions of acetone, and dried in adesiccator over KOH overnight. This process gave pure 15, but 6 and 11were purified on a 19×300 mm Prep Nova-Pak C18 column using a gradientof 2 to 20% CH₃CN in 0.1 M NH₄HCO₃.

Analytical reversed phase HPLC was carried out on a Waters 2965 systemwith a photodiode array detector, using an Atlantis C18 column, 100 Å,4.6 mm×50 mm, 3.0 μm. Gradients of CH₃CN and 0.1 M triethylammoniumacetate buffer (pH 6.8) were used with a flow rate of 1.0 mL/min. Lowresolution ESI-MS was routinely acquired using a Waters Micromass singlequadrupole LCZ system. LCMS of 6, 11, and 15 displayed m/z (M-H) 673(calculated for C₂₀H₂₃N₁₀O₁₃P₂ ⁻: 673).

Example 12 STING-Dependent Induction of Murine Alpha-Interferon andHuman CXCL10 by cGAMP Compounds

THP-1 Culture and Assay Conditions

THP-1 cells were cultured in RPMI1640 with 10% FBS, sodium pyruvate andpenicillin/streptomycin (Gibco, Life Technologies). 8×104 cells wereplated per 96-well in 100 μl of Medium and equilibrated for 2 h at 37°C./5% CO2. To generate macrophage-like cells, 8×104 THP-1 cells weredifferentiated overnight with 10 ng/ml PMA (Sigma), medium was changedand cells were incubated for additional 24 h prior to stimulation.

BMDM Culture and Assay Conditions

Bone marrow derived macrophage cells (BMDM) were flushed from femurs ofC57BL/6 mice. Erythrocytes were lysed (PharmLyse, BD Biosciences) and1×107 cells per Petri dish were incubated in DMEM 10% FBS, sodiumpyruvate and penicillin/streptomycin (Gibco, Life Technologies) with 30%L929-supernatant for 7 days. Cells were harvested with PBS 2 mM EDTA andplated at a density of 1×105 cells per 96-well. BMDMs weredigitonin-permeabilized or control-treated in the presence of indicatedcGAMP concentrations for 30 min, then supplemented with fresh medium.Supernatants were taken after 18 h and cytokines were determined byELISA.

Cell Permeabilization/Stimulation

Cell permeabilization for delivery of cyclic di-nucleotides wasperformed as previously described (Woodward et al., 2010). Briefly,supernatant was removed and cells were covered with 50 μl Perm-buffer(50 mM HEPES pH 7.0, 100 mM KCl, 3 mM MgCl2, 1 mM ATP, 0.1 mM GTP, 0.1mM DTT, 85 mM sucrose, 0.2% BSA)+/−10 μg/ml digitonin and serialdilutions of cGAMP isomers, followed by 30 min incubation at 37° C.Perm-buffer was then removed and cells were covered with 100 μl ofpre-warmed medium. Viability of permeabilized cells was >50% compared tountreated, as monitored by light microscopy and Cell Titer Blue (Roche).

Supernatants were collected 16 h after stimulation and cytokines weredetermined by ELISA and HEK-Blue™ IFN-α/β bioassay, respectively.

ELISA

Human CXCL-10 was determined by ELISA (BD Opteia human IP-10 ELISA-Set)according to manufacturer's recommendations. Murine Ifna was determinedby sandwich-ELISA: Monoclonal rat-anti Ifna (clone RMMA-1) was used ascapture antibody, recombinant Ifna was used as standard and polyclonalrabbit serum against Ifna for detection (all from PBL Interferon Source,Piscataway N.J., USA), followed by anti-rabbit HRP (Bio-Rad).

Fitting of Dose-Response Curves

4-parametric sigmoidal dose-response curves and EC50 values wereanalyzed with Graph Pad Prism (Graph Pad Software, La Jolla Calif.,USA).

TABLE S4 Proton and carbon chemical shifts for cGAMPs c[G(3′,5′)pAc[G(2′,5′)pA c[G(2′,5′)pA (3′,5′)p] (2′,5′)p] (3′,5′)p] Proton chemicalshifts list G H8 7.96 7.85 7.86 H1′ 5.92 5.99 5.93 H2′ 4.72 5.31 5.62H3′ 4.91 4.66 4.58 H4′ 4.39 4.45 4.39 H5′ 4.35 4.13 4.15 H5″ 4.07 4.214.22 A H8 8.35 8.21 8.31 H2 8.13 8.11 8.27 H1′ 6.11 6.29 6.17 H2′ 4.765.23 4.77 H3′ 4.92 4.69 5.03 H4′ 4.45 4.51 4.47 H5′ 4.38 4.16 4.45 H5″4.09 4.24 4.13 Carbon chemical shifts list G C8 139.72 142.36 143.29 C5118.97 119.88 120.06 C4 153.09 154.55 154.53 C1′ 92.23 89.13 88.90 C2′76.14 78.77 77.11 C3′ 73.18 73.87 74.05 C4′ 82.58 86.18 86.11 C5′ 65.0367.59 68.38 A C8 142.13 142.57 141.61 C6 157.97 157.89 157.97 C5 121.44121.10 121.47 C4 150.63 152.22 150.52 C2 155.32 155.08 155.05 C1′ 92.5687.63 92.36 C2′ 76.23 82.04 76.58 C3′ 73.16 74.75 73.17 C4′ 82.75 87.0382.68 C5′ 65.05 68.01 64.89

TABLE S5Primers used in the Quickchange mutagenesis PCRs (Metabion, Martinsried, D):Primer Name Sequence (5′-3′) N196A_Y200AcaaaggtgtggagcagctggccactggcagcgcctatgaacatgtgaagattN196A_Y200A_antisenseaatcttcacatgttcataggcgctgccagtggccagctgctccacacctttg K372Acctctctttctctcacactgaagcgtacattttgaataatcacggg K372A_antisensecccgtgattattcaaaatgtacgcttcagtgtgagagaaagagagg S165Attgaaacgcaaagatatcgcggaggcggccg S165A_antisensecggccgcctccgcgatatctttgcgtttcaa N172A ggcggccgagacggtggctaaagttgtggaacgcN172A_antisense gcgttccacaactttagccaccgtctcggccgcc Y200Agcagctgaacactggcagcgcctatgaacatgtgaagatt Y200A_antisenseaatcttcacatgttcataggcgctgccagtgttcagctgc R158A_R161AagaaggtgctggacaaattggcattgaaagccaaagatatctcggaggcggR158A_R161A_antisenseccgcctccgagatatctttggctttcaatgccaatttgtccagcaccttct K395Aaatcttccggagcaaaatgctgcagagcagaatgtttaaaattaatgaaatacc K395A_antisenseggtatttcattaattttaaacattctgctctgcagcattttgctccggaagatt R161Aggacaaattgagattgaaagccaaagatatctcggaggcg R161A_antisensecgcctccgagatatctttggctttcaatctcaatttgtcc G198P_S199Agtggagcagctgaacactgccgcctactatgaacatgtgaag G198P_S199A_antisensecttcacatgttcatagtaggcggcagtgttcagctgctccac G198Aggagcagctgaacactgccagctactatgaacatg G198A_antisensecatgttcatagtagctggcagtgttcagctgctcc G198Pgtggagcagctgaacactcccagctactatgaacatgt G198P_antisenseacatgttcatagtagctgggagtgttcagctgctccac G198P_G199Aggtgtggagcagctgaacactcccgcctactatgaacatgtgaagatt G198P_S199A_antisenseaatcttcacatgttcatagtaggcgggagtgttcagctgctccacacc S199Aggagcagctgaacactggcgcctactatgaacatgtgaag S199A_antisensecttcacatgttcatagtaggcgccagtgttcagctgctcc E211Atgtgaagatttctgctcctaatgcatttgatgttatgtttaaactgg E211A_antisenseccagtttaaacataacatcaaatgcattaggagcagaaatcttcaca K402AgcaaaatgctgcagaaaagaatgtttaaaattaatggcataccttttggaacagttgaaaaaaK402A_antisensettttttcaactgttccaaaaggtatgccattaattttaaacattcttttctgcagcattttgc S420Attcaagagctggatgcattctgtgcctaccatgtga S420A_antisensetcacatggtaggcacagaatgcatccagctcttgaa E371Agcctctctttctctcacactgcaaagtacattttgaataatcac E371A_antisensegtgattattcaaaatgtactttgcagtgtgagagaaagagaggc K424Agcattctgttcctaccatgtggcaactgccatctttcacatgtg K424A_antisensecacatgtgaaagatggcagttgccacatggtaggaacagaatgc R364Atcaaggagagacctgggccctctctttctctcac R364A_antisensegtgagagaaagagagggcccaggtctctccttga Y421Agctggatgcattctgttccgcccatgtgaaaactgccatc Y421A_antisensegatggcagttttcacatgggcggaacagaatgcatccagc mcGASfwXhoIatatatctcgagatggaagatccgcgtagaagga mcGASrevBglIIatatatagatctctatcaaagcttgtcaaaaattggaaacccat

TABLE S6 Oligonucleotides utilized for TLC analyses:Oligonucleotide Name Sequence (5′-3′) 45 mer DNA (top strand)tacagatctactagtgatctatgactgatctgtacatgatctaca 45 mer DNA (bottom strand)tgtagatcatgtacagatcagtcatagatcactagtagatctgta 45 mer RNA (top strand)uacagaucuacuagugaucuaugacugaucuguacaugaucuaca 45 mer RNA (bottom strand)uguagaucauguacagaucagucauagaucacuaguagaucugua 17 mer DNA (top strand)aaattgccgaagacgaa 17 mer DNA (bottom strand) tttcgtcttcggcaatt36 mer DNA (top strand) acacacacacacacacacacacacacacacacacac36 mer DNA (bottom strand) ctctctctctctctctctctctctctctctctctct

Bold and underlined nucleotides represent 8-oxoguanosines that wereutilized in separately generated modified oligos. Oligonucleotides weresynthesized in-house 3400 DNA synthesizer (Applied Biosystems) orpurchased (Dharmacon).

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Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

We claim:
 1. A compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: Ring A isselected from the group consisting of:

Ring B is selected from the group consisting of:

each X¹ and X² is independently —CH— or —N—; X³ is —NH—; X^(a) and X^(b)are independently —O— or —S—; X^(a1) and X^(b1) are —CH—; X^(c) andX^(d) are independently oxygen optionally substituted with cyanoethyl,or sulfur; each X^(e) and X^(f) is —O—; each W is independently P; eachR¹ and R² is independently selected from the group consisting ofhydrogen, halogen, —NH₂ and —OR^(a), wherein R^(a) is an oxygenprotecting group, hydrogen, or C₁₋₆ alkyl; each R³, R⁵, and R⁷ isindependently selected from the group consisting of hydrogen, halogen,—NH₂, —OR wherein R is hydrogen or C₁₋₆ alkyl, —SR wherein R is hydrogenor C₁₋₆ alkyl, and —NHC(O)R wherein R is hydrogen, C₁₋₆ alkyl, orphenyl; each R⁴ is independently selected from the group consisting ofhydrogen, halogen, —NH₂, —OR wherein R is C₁₋₆ alkyl, —SR wherein R ishydrogen or C₁₋₆ alkyl, and —NHC(O)R wherein R is hydrogen, C₁₋₆ alkyl,or phenyl; each R⁶ is independently selected from the group consistingof halogen, —NH₂, —OR wherein R is hydrogen or C₁₋₆ alkyl, —SR wherein Ris hydrogen or C₁₋₆ alkyl, and —NHC(O)R wherein R is hydrogen, C₁₋₆alkyl, or phenyl; each R¹⁰ and R¹¹ is independently hydrogen or C₁₋₂alkyl; and the subindex of the carbon atoms to which each R⁸ and R⁹ isattached is 0; with the proviso that the compound is other than:


2. The compound of claim 1, wherein Ring A is


3. The compound of claim 1, wherein Ring B is


4. The compound of claim 1, wherein Ring B is


5. The compound of claim 1, wherein R¹ is selected from the groupconsisting of hydrogen, halogen, and —OH.
 6. The compound of claim 5,wherein R² is selected from the group consisting of hydrogen, halogen,and —OH.
 7. The compound of claim 1, wherein the compound is of formulaII-a:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim1, wherein the compound is of formula X, XII, XIII, XIV, XV, or XVI:

or a pharmaceutically acceptable salt thereof.
 9. A compound:

or a pharmaceutically acceptable salt thereof.
 10. The compound of claim9, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim10, in isolated form.
 12. The compound of claim 11, in purified form.13. A pharmaceutical composition comprising the compound of claim 1 anda pharmaceutically acceptable carrier.
 14. The compound of claim 7,wherein Ring A is

each X¹ is N; R³ and R⁵ are hydrogen; R⁴ is NH₂; X^(a) and X^(b) are O;R¹ and R² are OH; and R¹⁰ and R¹¹ are hydrogen; or a pharmaceuticallyacceptable salt thereof.
 15. The compound of claim 14, wherein one orboth of X^(c) and X^(d) is sulfur.
 16. The compound of claim 14, whereinboth of X^(c) and X^(d) are oxygen.
 17. The compound of claim 14,wherein both of X^(c) and X^(d) are sulfur.
 18. The compound of claim 7,wherein Ring A is

each X¹ is N; R³ and R⁵ are hydrogen; R⁴ is NH₂; X^(a) and X^(b) are O;X^(c) and X^(d), are independently oxygen or sulfur; R¹ and R² are OH;and R¹⁰ and R¹¹ are hydrogen; or a pharmaceutically acceptable saltthereof.
 19. The compound of claim 1, wherein each X¹ is —N—.
 20. Thecompound of claim 1, wherein each X² is —N—.
 21. The compound of claim1, wherein X^(a) is —O—.
 22. The compound of claim 1, wherein X^(b) is—O—.
 23. The compound of claim 1, wherein X^(c) and X^(d) are bothoxygen.
 24. The compound of claim 1, wherein X^(c) and X^(d) are bothsulfur.
 25. The compound of claim 5, wherein R¹ is —OH.
 26. The compoundof claim 6, wherein R² is —OH.
 27. The compound of claim 1, wherein eachR³, R⁵, and R⁷ is independently selected from the group consisting ofhydrogen and halogen.
 28. The compound of claim 27, wherein each of R³,R⁵, and R⁷ is hydrogen.
 29. The compound of claim 1, wherein each of R⁴is independently selected from the group consisting of hydrogen,halogen, and —NH₂, and each of R⁶ is independently selected from thegroup consisting of halogen and —NH₂.
 30. The compound of claim 29,wherein R⁴ and R⁶ are each —NH₂.
 31. The compound of claim 1, whereinR¹⁰ and R¹¹ are hydrogen.
 32. The compound of claim 1, wherein each R³,R⁵, and R⁷ is independently selected from the group consisting ofhydrogen, halogen, —NH₂, —OR wherein R is hydrogen, —SR wherein R ishydrogen, and —NHC(O)R wherein R is hydrogen; each R⁴ is independentlyselected from the group consisting of hydrogen, halogen, —NH₂, —SRwherein R is hydrogen, and —NHC(O)R wherein R is hydrogen; each R⁶ isindependently selected from the group consisting of halogen, —NH₂, —ORwherein R is hydrogen, —SR wherein R is hydrogen, and —NHC(O)R wherein Ris hydrogen.
 33. A pharmaceutical composition comprising the compound ofclaim 7 and a pharmaceutically acceptable carrier.
 34. A pharmaceuticalcomposition comprising the compound of claim 17 and a pharmaceuticallyacceptable carrier.
 35. The compound of claim 1, wherein X^(c) and X^(d)are independently oxygen or sulfur.
 36. The compound of claim 1, whereineach R¹ and R² is independently selected from the group consisting ofhydrogen, halogen, —NH₂, and —OR^(a), wherein R^(a) is hydrogen or C₁₋₆alkyl.
 37. The compound of claim 1, wherein each R¹⁰ and R¹¹ ishydrogen.
 38. The compound of claim 1, wherein X^(c) and X^(d) areindependently oxygen or sulfur; and each R¹ and R² is independentlyselected from the group consisting of hydrogen, halogen, —NH₂, and—OR^(a), wherein R^(a) is hydrogen or C₁₋₆ alkyl.
 39. The compound ofclaim 1, wherein X^(c) and X^(d) are independently oxygen or sulfur; andeach R¹⁰ and R¹¹ is hydrogen.
 40. The compound of claim 1, wherein eachR¹ and R² is independently selected from the group consisting ofhydrogen, halogen, —NH₂, and —OR^(a), wherein R^(a) is hydrogen or C₁₋₆alkyl; and each R¹⁰ and R¹¹ is hydrogen.
 41. The compound of claim 1,wherein X^(c) and X^(d) are independently oxygen or sulfur; each R^(l)and R² is independently selected from the group consisting of hydrogen,halogen, —NH₂, and —OR^(a), wherein R^(a) is hydrogen or C₁₋₆ alkyl; andeach R¹⁰ and R¹¹ is hydrogen.
 42. The compound of claim 1, wherein thecompound is of formula XIV

wherein: R³ and R⁵ are independently hydrogen or halogen; R⁴ is selectedfrom the group consisting of hydrogen, halogen, and —NH₂; R⁶ is selectedfrom the group consisting of halogen and —NH₂; X^(c) and X^(d) areindependently O or S; R¹ is selected from the group consisting ofhydrogen, halogen, —OR^(a), and —NH₂, wherein R^(a) is hydrogen or C₁₋₆alkyl; and R² is selected from the group consisting of hydrogen,halogen, —OR^(a), and —NH₂, wherein R^(a) is hydrogen or C₁₋₆ alkyl. 43.The compound of claim 1, wherein the compound is of formula XVI

wherein: R³ and R⁵ are independently hydrogen or halogen; R⁴ is selectedfrom the group consisting of hydrogen, halogen, and —NH₂; X^(c) andX^(d) are independently O or S; R^(l) is selected from the groupconsisting of hydrogen, halogen, —OR^(a), and —NH₂, wherein R^(a) ishydrogen or C₁₋₆ alkyl; and R² is selected from the group consisting ofhydrogen, halogen, —OR^(a), and —NH₂, wherein R^(a) is hydrogen or C₁₋₆alkyl.
 44. A pharmaceutical composition comprising the compound of claim12 and a pharmaceutically acceptable carrier.